Compounds as CAV1.2 activators

Abstract

The invention provides a compound used as a CaV1.22 activating agent. In particular, a compound according to formula (I) or a pharmaceutically acceptable salt thereof is provided as a CaV1.22 activator for the treatment of schizophrenia, bipolar disorder, severe depressive disorder, substance use disorder, ADHD, Ferren-Mecdemedes syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Bruggea syndrome, short QT syndrome, and the like. Or early repolarization syndrome. # imgabs0 #

Description

[0001] This application is a divisional application of an international application with the application number PCT / IB2021 / 055183, titled "Methyl 2-(fluoromethyl)-5-oxo-4-phenyl-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate and methyl 2-(fluoromethyl)-5-oxo-4-phenyl-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate as CAV1.2 activators", which was filed on June 11, 2021. The international application entered the Chinese national phase on December 5, 2022, with the application number 202180040510.X. 1. Technical Field

[0002] The present invention relates to compounds of methyl 2-(fluoromethyl)-5-oxo-4-phenyl-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate and methyl 2-(fluoromethyl)-5-oxo-4-phenyl-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, pharmaceutical compositions containing them, and the use of such compounds as Ca V 1.2 activators for treating calcium signaling defects and / or synaptic dysfunction in the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada Syndrome, short QT syndrome, and early repolarization syndrome. 2. Background Art

[0003] Advances in human genomics have revealed the genetic basis of mental disorders. Genome-wide association studies (GWAS) of schizophrenia have identified more than a hundred disease-related loci, including Ca V 1.2 and other genes involved in neuronal calcium signaling. Cross-disorder GWAS analysis has identified Ca V 1.2 and its channel-forming β subunit (CACNB2) to be closely associated with schizophrenia, bipolar disorder, major depressive disorder, ADHD, and autism spectrum disorder. In addition to evidence from GWAS, exome sequencing of schizophrenia patients has shown an enrichment of disruptive mutations in Ca V 1.2 and other gene members of the neuronal calcium signaling pathway (including CACNA2D, CACNB, CAMK2 genes). Ca V1.2 has been shown to be important for neuronal differentiation and migration, neurite outgrowth, synaptic signaling, gene expression, and brain plasticity. It has been shown to play a role in mood, learning and memory, executive function, and the brain's reward response.

[0004] Ca V 1.2 is widely expressed throughout the body and plays a major role in multiple organ systems, including the cardiovascular system; however, Ca V 1.2 has different physiological functions in the cardiovascular system than in the brain. Studies have shown that Ca V 1.2 is a key factor in action potential generation in the heart, while it is a key driver of intracellular signaling and gene expression in neurons and has a minimal role in action potential generation. In Timothy Syndrome, the Ca V 1.2 mutation p.G406R results in different cellular phenotypes between cardiomyocytes and neurons. Ca V 1.2 mutations that cause cardiovascular-specific disorders (Brugada syndrome and long QT syndrome type 8) are further evidence that Ca V 1.2 has different functions in the heart and brain.

[0005] Calcium channel activators have been previously reported, but further research on their use in neuropsychiatric disorders has been limited due to their effects on the cardiovascular system. In fact, many of these molecules were initially studied and developed for their potential therapeutic use in heart failure. Most of the Ca V 1.2 SNPs associated with psychiatric GWAS studies are located in the introns of the gene, and these risk SNPs have been shown to be associated with a decrease in mRNA expression, which in many cases leads to an overall decrease in calcium current amplitude. Therefore, small molecules that can increase the overall current amplitude may be most beneficial to patients. 3. SUMMARY OF THE INVENTION

[0006] The present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof

[0007]

[0008] Wherein:

[0009] A is O or CH2;

[0010] R 1 is H or F;

[0011] R 2 is H or F; and

[0012] R 3is OCHF2, methyl, ethyl, or cyclopropyl, each of which is optionally substituted with 1 to 3 F.

[0013] In a second aspect, the present disclosure provides a compound according to formula (III), or a pharmaceutically acceptable salt or solvate thereof,

[0014]

[0015] wherein:

[0016] A is O or CH2;

[0017] R 1 is H or F;

[0018] R 2 is H or F;

[0019] R 3 is OCHF2, methyl, ethyl, or cyclopropyl, each of which is optionally substituted with 1 to 3 F; and

[0020] R 4 is H or F.

[0021] In a third aspect, the present disclosure provides a pharmaceutical composition comprising a compound having formula (I) or a pharmaceutically acceptable salt thereof.

[0022] In a fourth aspect, the present disclosure provides a compound having formula (I) or a pharmaceutically acceptable salt thereof for use in therapy, particularly in the treatment of the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada Syndrome, short QT syndrome, and early repolarization syndrome.

[0023] In a fifth aspect, the present disclosure provides a method for treating the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada syndrome, short QT syndrome, and early repolarization syndrome, the method comprising administering to a patient in need of treatment an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0024] In a sixth aspect, the present disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada Syndrome, short QT syndrome, and early repolarization syndrome.

[0025] In a seventh aspect, the present disclosure provides a method for treating the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada syndrome, short QT syndrome, and early repolarization syndrome, the method comprising administering to a patient in need of treatment a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0026] In an eighth aspect, the present disclosure provides a pharmaceutical composition comprising a compound of formula (III) or a pharmaceutically acceptable salt thereof.

[0027] In a ninth aspect, the present disclosure provides a compound of formula (III) or a pharmaceutically acceptable salt thereof for use in therapy, particularly for the treatment of the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada Syndrome, short QT syndrome, and early repolarization syndrome.

[0028] In a tenth aspect, the present disclosure provides a method for treating the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada Syndrome, short QT syndrome, and early repolarization syndrome, the method comprising administering to a patient in need of treatment an effective amount of a compound of formula (III) or a pharmaceutically acceptable salt thereof.

[0029] In an eleventh aspect, the present disclosure provides the use of a compound of formula (III) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada Syndrome, short QT syndrome, and early repolarization syndrome.

[0030] In a twelfth aspect, the present disclosure provides methods for treating the following diseases: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorder; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid Syndrome, and autism spectrum disorder; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada syndrome, short QT syndrome, and early repolarization syndrome, the method comprising administering to a patient in need of treatment a compound of formula (III) or a pharmaceutically acceptable salt thereof. 4. DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Shows the representative XRPD of Form A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate of the present disclosure. The x-axis shows the scattering angle (in °2-θ), and the y-axis shows the intensity of the scattered X-ray beam (in counts of detected photons).

[0032] Figure 2 : Shows the representative XRPD of Form B of [methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate of the present disclosure. The x-axis shows the scattering angle (in °2-θ), and the y-axis shows the intensity of the scattered X-ray beam (in counts of detected photons).

[0033] Figure 3 : Shows the representative XRPD of Form C of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate of the present disclosure of the present invention. The x-axis shows the scattering angle (in °2-θ), and the y-axis shows the intensity of the scattered X-ray beam (in counts of detected photons).

[0034] Figure 4 : Shows the representative XRPD of Hydrate A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate of the present disclosure. The x-axis shows the scattering angle (in °2-θ), and the y-axis shows the intensity of the scattered X-ray beam (in counts of detected photons).

[0035] Figure 5: Displays the representative XRPD of hydrate B of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate of the present disclosure. The x-axis shows the scattering angle (in °2-θ), and the y-axis shows the intensity of the scattered X-ray beam (in counts of detected photons).

[0036] Figure 6 : Displays a simulated cardiac action potential from the epicardial environment, which shows the effect of the voltage activation of Ca V 1.2 shifting to a more negative membrane potential. The x-axis is time (ms), and the y-axis is potential.

[0037] Figure 7 : Displays the single crystal X-ray of Example 6 (methyl (R)-4-(2-((S)-1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate), confirming that the active enantiomer is the R-enantiomer.

[0038] Figure 8 : Displays the single crystal X-ray of Example 8 (methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate), confirming that the active enantiomer is the R-enantiomer.

[0039] Figure 9 : Displays the single crystal X-ray of Example 9 (methyl (R)-4-(2-(difluoromethoxy)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate), confirming that the active enantiomer is the R-enantiomer. 5. Detailed Description

[0040] 5.1. Definitions

[0041] Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0042] As used herein, "administration (administering, administering, and administer)" refers to the manner of delivering the compounds described herein to a subject.

[0043] As used herein, "optionally substituted" means that the group mentioned may be substituted at one or more positions with any one or any combination of the groups listed hereinafter.

[0044] As used herein, "halo" or "halogen" may be fluorine, chlorine, bromine or iodine as used herein.

[0045] As used herein, "subject" refers to a living organism having one or more of the diseases or disorders described herein (e.g., mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders such as attention deficit hyperactivity disorder, Phelan-McDermid syndrome, and other autism spectrum disorders; neurodegenerative disorders such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions such as Brugada syndrome, short QT syndrome, and early repolarization syndrome) that can be treated by administering the pharmaceutical compositions described herein. Examples of subjects include mammals (e.g., humans and animals such as dogs, dairy cows, horses, monkeys, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals). In certain embodiments, the subject is a human, e.g., a human having, at risk of having, or likely to develop a disease described herein (e.g., mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders such as attention deficit hyperactivity disorder, Phelan-McDermid syndrome, and other autism spectrum disorders; neurodegenerative disorders such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions such as Brugada syndrome, short QT syndrome, and early repolarization syndrome).

[0046] Unless the context otherwise requires, throughout this specification and the claims which follow, the word "comprise" or variations such as "comprises" or "comprising" shall be understood to imply the inclusion of the stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0047] If there is a difference between the depicted structure and the chemical name given for that structure, the depicted structure shall prevail. Additionally, if the stereochemistry of a structure or a part of a structure is not indicated, for example, by thick or dashed lines, the structure or the part of the structure shall be interpreted to cover all stereoisomers of the structure or the part of the structure.

[0048] 5.2. Compounds

[0049] A compound according to formula (I), or a pharmaceutically acceptable salt or solvate thereof

[0050]

[0051] Wherein:

[0052] A is O or CH2;

[0053] R 1 is H or F;

[0054] R 2 is H or F; and

[0055] R 3 is OCHF2, methyl, ethyl, or cyclopropyl, each of which is optionally substituted with 1 to 3 Fs.

[0056] One embodiment is a compound having formula (Ia), or a pharmaceutically acceptable salt or solvate thereof,

[0057]

[0058] Another embodiment is a compound having formula (Ib), or a pharmaceutically acceptable salt or solvate thereof,

[0059]

[0060] In another embodiment, A is O.

[0061] In another embodiment, A is CH2.

[0062] In another embodiment, R 1 is H.

[0063] In another embodiment, R 1 is F.

[0064] In another embodiment, R 2 is H.

[0065] In another embodiment, R 2 is F.

[0066] In another embodiment, R 3 is OCHF2.

[0067] In another embodiment, R 3 is methyl, ethyl, or cyclopropyl, each of which is optionally substituted with 1 to 3 Fs.

[0068] In another embodiment, R 3 is methyl, ethyl, or cyclopropyl, each of which is substituted with 1 to 3 Fs.

[0069] In another embodiment, R 3 is CHF2 or CF3.

[0070] In another embodiment, R 3 is ethyl substituted with 1 or 2 F.

[0071] In another embodiment, R 3 is unsubstituted ethyl.

[0072] In another embodiment, R 3 is unsubstituted or cyclopropyl substituted with 2 F.

[0073] A compound according to formula (III), or a pharmaceutically acceptable salt or solvate thereof,

[0074]

[0075] wherein:

[0076] A is O or CH2;

[0077] R 1 is H or F;

[0078] R 2 is H or F;

[0079] R 3 is OCHF2, methyl, ethyl, or cyclopropyl, each of which is optionally substituted with 1 to 3 F; and

[0080] R 4 is H or F.

[0081] In another embodiment, A is O.

[0082] In another embodiment, A is CH2.

[0083] In another embodiment, R 1 is H.

[0084] In another embodiment, R 1 is F.

[0085] In another embodiment, R 2 is H.

[0086] In another embodiment, R 2 is F.

[0087] In another embodiment, R 3 is OCHF2.

[0088] In another embodiment, R 3 is methyl, ethyl, or cyclopropyl, each of which is optionally substituted with 1 to 3 F.

[0089] In another embodiment, R3 is methyl, ethyl, or cyclopropyl, each of which is substituted with 1 to 3 F.

[0090] In another embodiment, R 3 is CHF2 or CF3.

[0091] In another embodiment, R 3 is ethyl substituted with 1 or 2 F.

[0092] In another embodiment, R 3 is unsubstituted ethyl.

[0093] In another embodiment, R 3 is unsubstituted or cyclopropyl substituted with 2 F.

[0094] In another embodiment, R 4 is F.

[0095] In another embodiment, R 4 is H.

[0096] Specific compounds include:

[0097] Methyl (R)-4-(2-((R)-2,2-difluorocyclopropyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0098] Methyl (R)-4-(2-((S)-2,2-difluorocyclopropyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0099] (R)-4-(2-cyclopropyl-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0100] Methyl (R)-4-(2-((R)-2,2-difluorocyclopropyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0101] Methyl (R)-4-(2-((S)-2,2-difluorocyclopropyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0102] Methyl (R)-4-(3,5-difluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0103] Methyl (R)-4-(3,5-difluoro-2-((S)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0104] Methyl (R)-4-(2-((S)-1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0105] Methyl (R)-4-(2-((R)-1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate;

[0106] Methyl (R)-4-(2-((S)-1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate;

[0107] Methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0108] Methyl (R)-4-(3-fluoro-2-((S)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0109] Methyl (R)-4-(2-(difluoromethoxy)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0110] Methyl (R)-4-(3,5-difluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0111] Methyl (R)-4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0112] Methyl (R)-4-(2-(2,2-difluoroethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0113] Methyl (R)-4-(2-cyclopropyl-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0114] Methyl (R)-4-(2-cyclopropyl-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate;

[0115] Methyl (R)-4-(2-(difluoromethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0116] Methyl (R)-4-(2-((R)-1,2-difluoroethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0117] Methyl (R)-4-(2-((S)-1,2-difluoroethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0118] Methyl (R)-4-(3-fluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0119] Methyl (R)-4-(2-(difluoromethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0120] Methyl (R)-4-(2-(difluoromethoxy)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0121] Methyl 4-(2-(difluoromethoxy)-5-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0122] Methyl (R)-4-(2-cyclopropyl-5-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0123] Methyl (R)-4-(2-cyclopropylphenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0124] Methyl (R)-4-(5-fluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0125] Methyl (R)-2-(fluoromethyl)-5-oxo-4-(2-(trifluoromethyl)phenyl)-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate;

[0126] (R)-4-(3-fluoro-2-(2,2,2-trifluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate;

[0127] Methyl (R)-4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate;

[0128] Methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate;

[0129] Methyl (R)-4-(3-fluoro-2-((S)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate;

[0130] Methyl (R)-4-(2-cyclopropyl-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate;

[0131] Methyl (S)-4-(3,5-difluoro-2-((S)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate; and

[0132] Methyl (S)-4-(3,5-difluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate.

[0133] The specific compounds further include:

[0134] Methyl (R)-4-(2-ethylphenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate; and

[0135] Methyl (R)-2-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate.

[0136] Depending on the choice of starting materials and procedures, the compounds can exist in possible stereoisomeric forms or as mixtures thereof (e.g., as pure optical isomers or as mixtures of stereoisomers such as racemates and mixtures of diastereomers), depending on the number of asymmetric carbon atoms. This disclosure is intended to include all such possible stereoisomers, including racemic mixtures, mixtures of diastereomers, and optically pure forms. The optically active (R)- and (S)-stereoisomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituents can be in the E or Z configuration. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents can have cis or trans configurations. All tautomeric forms are also intended to be included.

[0137] As used herein, the term "salt(s)" refers to acid addition salts or base addition salts of the compounds of this disclosure. "Salt" particularly includes "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of this disclosure and are typically not biologically or otherwise undesirable. In many cases, due to the presence of amino and / or carboxyl groups or groups similar thereto, the compounds of this disclosure are capable of forming acid salts and / or base salts.

[0138] Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.

[0139] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.

[0140] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, etc.

[0141] Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.

[0142] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the Periodic Table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts.

[0143] Organic bases from which salts can be derived include, for example, primary amines, secondary amines, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; basic ion exchange resins, etc. Certain organic amines include isopropylamine, benzathine, choline salts, diethanolamine, diethylamine, lysine, glucosamine, piperazine, and tromethamine.

[0144] In another aspect, the present disclosure provides the compounds of the present disclosure in the form of: acetate, ascorbate, adipate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, caprate, chloride / hydrochloride, chlortheophyllonate, citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide / iodide, hydroxyethylsulfonate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methyl sulfate, mucate, naphthoate, naphthalenesulfonate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / monohydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, sebacate, stearate, succinate, sulfosalicylate, sulfate, tartrate, tosylate, trifenatate, trifluoroacetate, or cinchomeronate.

[0145] The compounds of the present disclosure, i.e., compounds of formula (I) containing groups capable of acting as hydrogen bond donors and / or acceptors, are capable of forming co-crystals with suitable co-crystal formers. These co-crystals can be prepared from the compounds of formula (I) by known co-crystal formation procedures. Such procedures include grinding, heating, co-sublimation, co-melting, or contacting the compounds of formula (I) with co-crystal formers in solution under crystallization conditions, and separating the co-crystals thus formed. Suitable co-crystal formers include those described in WO 2004 / 078163. Accordingly, the present disclosure further provides co-crystals comprising the compounds of formula (I).

[0146] In addition, the compounds of the present disclosure, including their salts, may also be obtained in the form of their hydrates, or include other solvents used for their crystallization. The compounds of the present disclosure may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); thus, it is intended that the present disclosure includes both solvated and non-solvated forms. The term "solvate" refers to a molecular complex of a compound of the present disclosure (including its pharmaceutically acceptable salts) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical field and are known to be harmless to recipients, such as water, ethanol, etc. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0147] The present disclosure includes both unlabeled and isotopically labeled forms of the compounds having formula (I). The isotopically labeled compounds have the structures depicted by the formulae given herein, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Isotopes that may be incorporated into the compounds of the present disclosure include, for example, isotopes of hydrogen.

[0148] In addition, incorporation of certain isotopes, particularly deuterium (i.e., 2 H or D) can provide certain therapeutic advantages resulting from increased metabolic stability, such as increased in vivo half-life or reduced dose requirements or improved therapeutic index or tolerance. It should be understood that deuterium is considered a substituent of the compounds of the present disclosure in this context. The concentration of deuterium can be defined by the isotope enrichment factor. As used herein, the term "isotope enrichment factor" means the ratio of the isotope abundance to the natural abundance of the designated isotope. If a substituent in a compound of the present disclosure is indicated as deuterium, then such a compound has an isotope enrichment factor of at least 3500 (52.5% deuterium incorporation on each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation) for each designated deuterium atom. It should be understood that the term "isotope enrichment factor" can be applied to any isotope in the same manner as described for deuterium.

[0149] For example, deuteration of formula (I) is shown as formula (II):

[0150]

[0151] or its pharmaceutically acceptable salt, wherein R 1 、R 2 、R 3 、and A are as defined in formula (I); RD1 to R D8 are each independently H or D.

[0152] Other examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as 3 H, 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl, 123 I, 124 I, 125 I. Thus, it should be understood that the present disclosure includes compounds incorporating one or more of any of the foregoing isotopes (including, for example, radioactive isotopes such as 3 H and 14 C), or those in which non-radioactive isotopes such as 2 H and 13 C are present. Such isotopically labeled compounds can be used in metabolic studies (with 14 C), reaction kinetics studies (e.g., with 2 H or 3 H), detection or imaging techniques (such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including determination of drug or substrate tissue distribution), or for radioactive treatment of patients. In particular, 18 F or labeled compounds may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds of the present disclosure can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the appended examples and preparations, using appropriate isotopically labeled reagents in place of the unlabeled reagents previously used.

[0153] Any asymmetric atoms (such as carbon, etc.) of one or more compounds of the present disclosure can exist in a racemic or enantiomerically enriched configuration, e.g., (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom has an (R)- or (S)-configuration with an enantiomeric excess of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. If possible, substituents on atoms with unsaturated double bonds can exist in the cis-(Z)- or trans-(E)-form.

[0154] Accordingly, as used herein, the compounds of the present disclosure can be in the form of one of the possible stereoisomers, rotamers, atropisomers, tautomers, or mixtures thereof, e.g., as substantially pure geometric (cis or trans) stereoisomers, diastereomers, optical isomers (enantiomers), racemates, or mixtures thereof.

[0155] Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric or optical isomers, diastereomers, racemates based on the physicochemical differences of the components, e.g., by chromatography and / or fractional crystallization.

[0156] The racemates of any resulting compounds or intermediates of the present disclosure can be resolved into the optically active enantiomers by known methods, e.g., by separating the diastereomeric salts obtained with an optically active acid or base and liberating the optically active acidic or basic compound. In particular, the compounds of the present disclosure can thus be resolved into their optical enantiomers using a basic moiety, e.g., by fractional crystallization of salts formed with the following optically active acids, e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di - O,O'-p - toluoyl tartaric acid, mandelic acid, malic acid, or camphor - 10 - sulfonic acid. The racemic compounds or racemic intermediates of the present disclosure can also be resolved by chiral chromatography (e.g., high - performance liquid chromatography (HPLC) using a chiral adsorbent).

[0157] Method of Use

[0158] The compounds of the present disclosure, in free form or in the form of a pharmaceutically acceptable salt, exhibit valuable pharmacological properties, e.g., Ca V 1.2 activation properties, e.g., as shown by the in vitro and in vivo tests provided in the next section, and thus are indicated for therapy or for use as research chemicals, e.g., as tool compounds.

[0159] The compounds of the present disclosure can be used for the treatment of indications selected from the following list: mental disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders;

[0160] neurodevelopmental disorders such as attention - deficit / hyperactivity disorder, Phelan - McDermid Syndrome, and autism spectrum disorder; and

[0161] neurodegenerative disorders such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease, and

[0162] Cardiac disorders, such as Brugada syndrome, short QT syndrome, and early repolarization syndrome. In one embodiment, the indication is a mental disorder, such as schizophrenia, bipolar disorder, major depressive disorder, and substance use disorder. In another embodiment, the indication is schizophrenia or bipolar disorder.

[0163] Accordingly, as a further aspect, the present disclosure provides the use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in therapy. In a further embodiment, the therapy is selected from diseases that can be treated by activating Ca V v1.2. In another embodiment, the disease is selected from the list of indications above.

[0164] Accordingly, as a further aspect, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt thereof for use in therapy. In a further embodiment, the therapy is selected from diseases that can be treated by activating Ca V v1.2. In another embodiment, the disease is selected from the list of indications above.

[0165] In another aspect, the present disclosure provides a method for treating a disease that can be treated by activating Ca V v1.2, the method comprising administering a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof. In a further embodiment, the disease is selected from the list of indications above.

[0166] In another aspect, the present disclosure provides a method for treating a disease that can be treated by activating Ca V v1.2, the method comprising administering a compound of the present disclosure or a pharmaceutically acceptable salt thereof. In a further embodiment, the disease is selected from the list of indications above.

[0167] Accordingly, as a further aspect, the present disclosure provides the use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the manufacture of a medicament. In a further embodiment, the medicament is for treating a disease that can be treated by activating Ca V v1.2. In another embodiment, the disease is selected from the list of indications above.

[0168] In another aspect, the present disclosure provides a method for treating schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome that can be treated by activating Ca V v1.2, the method comprising administering a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof.

[0169] In another aspect, the present disclosure provides a method for treating schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome, which is treated by activating Ca V 1.2, the method comprising administering a compound of the present disclosure or a pharmaceutically acceptable salt thereof.

[0170] Accordingly, as a further aspect, the present disclosure provides the use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment of a disease selected from the group consisting of: schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome.

[0171] Accordingly, as a further aspect, the present disclosure provides the use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment of a disease selected from the group consisting of: schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome.

[0172] In another aspect, the present disclosure provides a method for treating schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome, the method comprising administering a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof.

[0173] In another aspect, the present disclosure provides a method for treating schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome, the method comprising administering a compound of the present disclosure or a pharmaceutically acceptable salt thereof.

[0174] Accordingly, as a further aspect, the present disclosure provides the use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome.

[0175] Drug composition

[0176] In another aspect, the present disclosure provides a drug composition comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In additional embodiments, the composition comprises at least two pharmaceutically acceptable carriers (such as those described herein). The drug composition can be formulated for a specific route of administration, such as oral administration, parenteral administration (such as by injection, infusion, transdermal or topical administration), and rectal administration. Topical administration can also involve inhalation or intranasal application. The drug compositions of the present disclosure can be made in solid form (including but not limited to capsules, tablets, pills, granules, powders or suppositories), or in liquid form (including but not limited to solutions, suspensions or emulsions). Tablets can be film-coated or enteric-coated according to methods known in the art. Typically, the drug composition is a tablet or gelatin capsule comprising the active ingredient and one or more of the following:

[0177] a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine;

[0178] b) lubricants, such as silica, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; also included for tablets

[0179] c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if desired

[0180] d) disintegrants, such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and

[0181] e) adsorbents, colorants, flavorants and sweeteners.

[0182] For a subject of about 50 - 70 kg, the drug composition or combination of the present disclosure can be a unit dose of one or more active ingredients of about 1 - 1000 mg, or a unit dose of the active ingredient of about 1 - 500 mg or about 1 - 250 mg or about 1 - 150 mg or about 0.5 - 100 mg, or about 1 - 50 mg. The therapeutically effective dose of the compound, drug composition, or combination thereof depends on the species, body weight, age of the subject and the individual condition, disorder or disease being treated or its severity. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each active ingredient necessary to prevent, treat or inhibit the progression of a disorder or disease.

[0183] Use favorable mammals (e.g., mice, rats, dogs, monkeys) or their isolated organs, tissues, and products to demonstrate the above dose characteristics in in vitro and in vivo tests. The compounds of the present disclosure can be applied in vitro in solution form (e.g., aqueous solution), and can be applied in vivo enterally, parenterally, advantageously intravenously, e.g., as a suspension or in an aqueous solution. The in vitro dose can be in the range between about 10 -3 molar concentration and 10 -9 molar concentration. Depending on the route of administration, the therapeutically effective amount in vivo can be in the range between about 0.1 - 500 mg / kg, or between about 1 - 100 mg / kg.

[0184] Crystalline form

[0185] The present disclosure relates to a crystalline form of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, and to a method for its preparation. Furthermore, the present disclosure relates to a pharmaceutical composition comprising preferably a predetermined amount and / or an effective amount of the crystalline form of said methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate and at least one pharmaceutically acceptable excipient. The pharmaceutical composition of the present disclosure can be used as a medicine, particularly for treating and / or preventing schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome.

[0186] Different solid forms of an active pharmaceutical ingredient generally have different properties. Differences in the physicochemical properties of the solid forms can play a key role in the improvement of pharmaceutical compositions. For example, due to the improvement of the solid form of the active pharmaceutical ingredient, pharmaceutical formulations with improved dissolution profiles or improved stability or shelf life can become readily available. It is also possible to improve the processing or handling of the active pharmaceutical ingredient during the formulation process. The new solid forms of the active pharmaceutical ingredient can thus have desirable processing properties. Compared with previously known solid forms, they are easier to handle, more suitable for storage and / or allow better purification.

[0187] The crystalline forms of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate can be characterized by analytical methods well known in the pharmaceutical industry for the characterization of solids. Such methods include, but are not limited to, XRPD, SXRD, FTIR, Raman, DSC, TGA, and GMS (gravimetric measurement system). These forms can be characterized by one of the aforementioned analytical methods or by combining two or more of them.

[0188] The crystalline forms of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate can be referred to herein as being characterized by graphical data "as shown in the figures". Such data includes, for example, powder X-ray diffraction, FTIR, and Raman spectroscopy. Those skilled in the art will appreciate that factors such as variations in instrument type, response, and sample orientation, sample concentration, and sample purity may result in small variations in such data when presented in graphical form, such as variations related to precise peak positions and intensities. However, comparison of the graphical data in the figures herein with graphical data generated for another or unknown solid form and confirmation that the two sets of graphical data pertain to the same crystal form are well within the knowledge of those skilled in the art.

[0189] As used herein, the term "reflection" with respect to powder X-ray diffraction means a peak in the X-ray diffraction pattern that is caused by constructive interference of X-rays scattered by parallel planes of atoms in a solid material, the solid material being distributed in a sequential and repeating pattern in long-range positional order. Such a solid material is classified as a crystalline material, while an amorphous material is defined as a solid material that lacks long-range order and only exhibits short-range order, thus resulting in broad scattering. According to the literature, long-range order extends, for example, over approximately 100 to 1000 atoms, while short-range order extends only over a few atoms (see Fundamentals of Powder Diffraction and Structural Characterization of Materials by Vitalij K. Pecharsky and Peter Y. Zavalij, Kluwer Academic Publishers, 2003, page 3).

[0190] The term "substantially the same" with respect to powder X-ray diffraction means that the reflection positions and the variability of the relative intensities of the reflections need to be considered. For example, the typical accuracy of the 2-θ value is in the range of ±0.2° 2-θ, preferably in the range of ±0.1° 2-θ.

[0191] In addition, those skilled in the art will understand that the relative reflection intensities will show variability between devices and variability due to crystallinity, preferred orientation, particle size, sample preparation, and other factors known to those skilled in the art, and should only be used as a qualitative measurement.

[0192] The terms "solid form" or "solid state form" as used interchangeably herein refer to any crystalline and / or amorphous phase of a compound.

[0193] As used herein, the term "amorphous" refers to a solid form of a compound that is non-crystalline. An amorphous compound does not have long-range order and does not exhibit a definitive X-ray diffraction pattern with reflections.

[0194] As used herein, the term "polymorph" refers to a crystalline form having the same chemical composition but a different spatial arrangement of molecules, atoms, and / or ions that form the crystal.

[0195] As used herein, the term "cocrystal" refers to a crystalline material containing two or more different molecular and / or ionic compounds associated by non-ionic and non-covalent bonds within the same crystal lattice, where at least two individual molecular and / or ionic compounds are solids at room temperature.

[0196] As used herein, the term "hydrate" refers to a crystalline solid in which water is incorporated into or accommodated by (e.g., as part of) the crystal structure or trapped within the crystal (water inclusion). Thus, water can be present in stoichiometric or non-stoichiometric amounts. When water is present in stoichiometric amounts, the hydrate can be referred to by adding a Greek numerical prefix. For example, a hydrate can be called a hemihydrate or a monohydrate, depending on the water / compound stoichiometry. The water content can be measured, for example, by Karl-Fischer-Coulometry.

[0197] As used herein, the term "dehydrating or dehydration" describes the removal of at least part of the water from the crystal structure of the host molecule.

[0198] As used herein, the term "solvate" refers to a crystalline solid in which one or more organic solvents are incorporated within or entrapped by the crystal structure (e.g., as part of the crystal structure) or occluded in the crystal (water inclusion). Thus, one or more organic solvents can be present in stoichiometric or non-stoichiometric amounts. When one or more organic solvents are present in one or more stoichiometric amounts, the solvate can be designated by adding a Greek numerical prefix. For example, depending on the stoichiometry of the one or more solvents / compounds, the solvate can be termed a hemisolvate or a monosolvate. The solvent content can be measured, for example, by GC, NMR, SXRD, and / or TGA / MS.

[0199] As used herein, the term "non-hygroscopic" refers to a compound that exhibits a water uptake of up to 2 w-% based on the weight of the compound in an adsorption cycle at relative humidities in the range from 0 to 95% RH and at a temperature of (25.0 ± 0.1) °C as measured by GMS.

[0200] As used herein, the term "about" means within a range that is statistically meaningful. Such a range can be within one order of magnitude of the indicated value or range, typically within 10%, more typically within 5%, even more typically within 1%, and most typically within 0.1%. Sometimes, such a range can be within the experimental error, which is typical of standard methods for measuring and / or determining a given value or range.

[0201] As used herein, with respect to a composition comprising a specific solid form of (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, the term "substantially free of any other solid form" means that the composition comprises up to 20 w-%, preferably up to 10 w-%, more preferably up to 5 w-%, even more preferably up to 2 w-%, and most preferably up to 1 w-% of any other solid form of (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, based on the weight of the composition.

[0202] As used herein, when used in reference to a form, "substantially pure" means a compound having a purity greater than 90 w-% based on the weight of the compound, including greater than 90 w-%, 91 w-%, 92 w-%, 93 w-%, 94 w-%, 95 w-%, 96 w-%, 97 w-%, 98 w-%, and 99 w-%, and also including a purity equal to about 100 w-% of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. The remaining material comprises one or more other forms of the compound, and / or reaction impurities and / or processing impurities resulting from its preparation. For example, a crystalline form of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate can be considered substantially pure because it has a purity greater than 90 w-% as measured by means known and generally accepted in the art at this time, where the remaining material less than 10 w-% comprises one or more other forms of [Compound ABC] and / or reaction impurities and / or processing impurities.

[0203] Crystalline Form A

[0204] In another aspect, the present disclosure provides crystalline Form A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. Table 1 shows the most prominent X-ray powder diffraction peaks of Form A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate.

[0205] Table 1. Most Prominent X-ray Powder Diffraction Peaks of Form A

[0206]

[0207] One embodiment of the present disclosure provides Form A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which is characterized in that: when measured at a temperature in the range of 20 °C to 30 °C with Cu-Kα 1,2 radiation having a wavelength of 0.15419 nm, it has an XRPD pattern comprising reflections at the following 2-θ angles:

[0208] (8.5 ± 0.2)°, (10.4 ± 0.2)°, and (10.8 ± 0.2)°; or

[0209] (8.5 ± 0.2)°, (10.4 ± 0.2)°, (10.8 ± 0.2)°, and (22.4 ± 0.2)°; or

[0210] (8.5 ± 0.2)°, (10.4 ± 0.2)°, (10.8 ± 0.2)°, (14.4 ± 0.2)°, and (22.4 ± 0.2)°; or

[0211] (8.5 ± 0.2)°, (10.4 ± 0.2)°, (10.8 ± 0.2)°, (14.4 ± 0.2)°, (21.6 ± 0.2)°, and (22.4 ± 0.2)°; or

[0212] (7.2 ± 0.2)°, (8.5 ± 0.2)°, (10.4 ± 0.2)°, (10.8 ± 0.2)°, (14.4 ± 0.2)°, (21.6 ± 0.2)°, and (22.4 ± 0.2)°; or

[0213] (7.2 ± 0.2)°, (8.5 ± 0.2)°, (10.4 ± 0.2)°, (10.8 ± 0.2)°, (14.4 ± 0.2)°, (18.5 ± 0.2)°, (21.6 ± 0.2)°, and (22.4 ± 0.2)°; or

[0214] (7.2 ± 0.2)°, (8.5 ± 0.2)°, (10.4 ± 0.2)°, (10.8 ± 0.2)°, (14.4 ± 0.2)°, (15.6 ± 0.2)°, (18.5 ± 0.2)°, (21.6 ± 0.2)°, and (22.4 ± 0.2)°; or

[0215] (6.7 ± 0.2)°, (7.2 ± 0.2)°, (8.5 ± 0.2)°, (10.4 ± 0.2)°, (10.8 ± 0.2)°, (14.4 ± 0.2)°, (15.6 ± 0.2)°, (18.5 ± 0.2)°, (21.6 ± 0.2)°, and (22.4 ± 0.2)°; or

[0216] (6.7 ± 0.2)°, (7.2 ± 0.2)°, (8.5 ± 0.2)°, (10.4 ± 0.2)°, (10.8 ± 0.2)°, (13.5 ± 0.2)°, (14.4 ± 0.2)°, (15.6 ± 0.2)°, (18.5 ± 0.2)°, (21.6 ± 0.2)°, and (22.4 ± 0.2)°.

[0217] In yet another embodiment, the present disclosure relates to a crystalline form (Form A) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which crystalline form is characterized by having an XRPD pattern substantially the same as that shown in the present disclosure when measured at a temperature in the range of 20 °C to 30 °C with Cu-Kα radiation having a wavelength of 0.15418 nm. 1,2 when measured with Figure 1 the XRPD pattern shown in the present disclosure.

[0218] In another embodiment, the present disclosure relates to a crystalline form (Form A) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which crystalline form is characterized by having an onset melting temperature of 98 °C ± 5 °C when measured by DSC at a heating rate of 10 K / min.

[0219] In another embodiment, the present disclosure relates to a crystalline form (Form A) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which crystalline form is characterized by having a peak melting temperature of 104 °C ± 5 °C when measured by DSC at a heating rate of 10 K / min.

[0220] Crystalline Form B

[0221] In a further aspect, the present disclosure provides Form B of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. Table 2 shows the most prominent X-ray powder diffraction peaks of Form B of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate.

[0222] Table 2. Most Prominent X-ray Powder Diffraction Peaks of Form B

[0223]

[0224] In one embodiment, the present disclosure provides Form B of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which is characterized in that when measured at a temperature in the range of 20 °C to 30 °C with Cu-Kα radiation having a wavelength of 0.15419 nm 1,2 it has an XRPD pattern having reflections at the following 2-θ angles:

[0225] (12.0 ± 0.2)°, (13.7 ± 0.2)°, and (21.4 ± 0.2)°; or

[0226] (12.0 ± 0.2)°, (13.7 ± 0.2)°, (18.3 ± 0.2)°, and (21.4 ± 0.2)°; or

[0227] (12.0 ± 0.2)°, (13.7 ± 0.2)°, (18.3 ± 0.2)°, (21.4 ± 0.2)°, and (23.8 ± 0.2)°; or

[0228] (12.0 ± 0.2)°, (13.7 ± 0.2)°, (18.3 ± 0.2)°, (21.4 ± 0.2)°, (23.0 ± 0.2)°, and (23.8 ± 0.2)°; or

[0229] (9.1 ± 0.2)°, (12.0 ± 0.2)°, (13.7 ± 0.2)°, (18.3 ± 0.2)°, (21.4 ± 0.2)°, (23.0 ± 0.2)°, and (23.8 ± 0.2)°; or

[0230] (9.1 ± 0.2)°, (12.0 ± 0.2)°, (13.7 ± 0.2)°, (14.5 ± 0.2)°, (18.3 ± 0.2)°, (21.4 ± 0.2)°, (23.0 ± 0.2)°, and (23.8 ± 0.2)°; or

[0231] (9.1 ± 0.2)°, (12.0 ± 0.2)°, (13.7 ± 0.2)°, (14.5 ± 0.2)°, (15.7 ± 0.2)°, (18.3 ± 0.2)°, (21.4 ± 0.2)°, (23.0 ± 0.2)°, and (23.8 ± 0.2)°.

[0232] In yet another embodiment, the present disclosure relates to a crystalline form (Form B) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which crystalline form is characterized by having an XRPD pattern substantially the same as that shown in the present invention when measured at a temperature in the range of 20 °C to 30 °C with Cu-Kα radiation having a wavelength of 0.15418 nm. 1,2 when measured with Figure 2 an XRPD pattern substantially the same as that shown in the present invention.

[0233] In another embodiment, the present disclosure relates to a crystalline form (Form B) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which crystalline form is characterized by having a melting onset temperature of 184 °C ± 5 °C when measured by DSC at a heating rate of 10 K / min.

[0234] In another embodiment, the present disclosure relates to a crystalline form (Form B) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which crystalline form is characterized by having a melting peak maximum temperature of 185 °C ± 5 °C when measured by DSC at a heating rate of 10 K / min.

[0235] Crystalline Form C

[0236] In a further embodiment, the present disclosure provides Form C of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. Table 3 shows the most prominent X-ray powder diffraction peaks of Form C of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate.

[0237] Table 3. Most prominent X-ray powder diffraction peaks of Form C

[0238]

[0239] In one embodiment, the present disclosure provides Form C of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which is characterized in that when measured at a temperature in the range of 20 °C to 30 °C with Cu-Kα radiation having a wavelength of 0.15419 nm 1,2 it has an XRPD pattern having reflections at the following 2-θ angles:

[0240] (10.3 ± 0.2)°, (15.5 ± 0.2)°, and (20.6 ± 0.2)°; or

[0241] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (15.5 ± 0.2)°, and (20.6 ± 0.2)°; or

[0242] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (15.5 ± 0.2)°, (20.6 ± 0.2)°, and (22.7 ± 0.2)°; or

[0243] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (15.5 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, and (22.7 ± 0.2)°; or

[0244] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (15.5 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, (22.7 ± 0.2)°, and (22.0 ± 0.2)°; or

[0245] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (15.5 ± 0.2)°, (16.6 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, (22.7 ± 0.2)°, and (22.0 ± 0.2)°; or

[0246] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (12.8 ± 0.2)°, (15.5 ± 0.2)°, (16.6 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, (22.7 ± 0.2)°, and (22.0 ± 0.2)°; or

[0247] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (12.8 ± 0.2)°, (15.5 ± 0.2)°, (16.6 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, (22.7 ± 0.2)°, (22.0 ± 0.2)° and (23.2 ± 0.2)°; or

[0248] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (12.3 ± 0.2)°, (12.8 ± 0.2)°, (15.5 ± 0.2)°, (16.6 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, (22.7 ± 0.2)°, (22.0 ± 0.2)° and (23.2 ± 0.2)°; or

[0249] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (12.3 ± 0.2)°, (12.8 ± 0.2)°, (15.5 ± 0.2)°, (16.6 ± 0.2)°, (17.3 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, (22.7 ± 0.2)°, (22.0 ± 0.2)° and (23.2 ± 0.2)°; or

[0250] (10.3 ± 0.2)°, (11.3 ± 0.2)°, (12.3 ± 0.2)°, (12.8 ± 0.2)°, (14.0 ± 0.2)°, (15.5 ± 0.2)°, (16.6 ± 0.2)°, (17.3 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, (22.7 ± 0.2)°, (22.0 ± 0.2)° and (23.2 ± 0.2)°; or

[0251] (7.0 ± 0.2)°, (10.3 ± 0.2)°, (11.3 ± 0.2)°, (12.3 ± 0.2)°, (12.8 ± 0.2)°, (14.0 ± 0.2)°, (15.5 ± 0.2)°, (16.6 ± 0.2)°, (17.3 ± 0.2)°, (18.2 ± 0.2)°, (20.6 ± 0.2)°, (22.7 ± 0.2)°, (22.0 ± 0.2)° and (23.2 ± 0.2)°.

[0252] In yet another embodiment, the present disclosure relates to a crystalline form (Form C) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which crystalline form is characterized by having the following X-ray powder diffraction pattern when measured at a temperature in the range of 20 °C to 30 °C using Cu-Kα radiation having a wavelength of 0.15418 nm1,2 During radiation measurement, it has an XRPD pattern that is substantially the same as that shown in the present invention. Figure 3 shown.

[0253] In another embodiment, the present disclosure relates to a crystalline form (Form C) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which is characterized by a melting onset temperature of 178 °C ± 5 °C when measured by DSC at a heating rate of 10 K / min.

[0254] In one embodiment, the present disclosure relates to a crystalline form (Form C) of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which is characterized by a highest melting peak temperature of 178 °C ± 5 °C when measured by DSC at a heating rate of 10 K / min.

[0255] Hydrate A

[0256] In a further aspect, the present disclosure provides hydrate A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. Table 4 shows the most prominent X-ray powder diffraction peaks of the crystalline form hydrate A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate.

[0257] Table 4. Most prominent X-ray powder diffraction peaks of hydrate A

[0258]

[0259] In one embodiment, the present disclosure provides hydrate A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which is characterized by an XPRD pattern having reflections at the following 2-θ angles:

[0260] (10.0 ± 0.2)°, (10.6 ± 0.2)° and (23.5 ± 0.2)°; or

[0261] (10.0 ± 0.2)°, (10.6 ± 0.2)°, (23.5 ± 0.2)° and (25.0 ± 0.2)°; or

[0262] (10.0 ± 0.2)°, (10.6 ± 0.2)°, (14.4 ± 0.2)°, (23.5 ± 0.2)° and (25.0 ± 0.2)°; or (10.0 ± 0.2)°, (10.6 ± 0.2)°, (14.4 ± 0.2)°, (22.9 ± 0.2)°, (23.5 ± 0.2)° and (25.0 ± 0.2)°; or

[0263] (10.0 ± 0.2)°, (10.6 ± 0.2)°, (12.8 ± 0.2)°, (14.4 ± 0.2)°, (22.9 ± 0.2)°, (23.5 ± 0.2)° and (25.0 ± 0.2)°; or

[0264] (10.0 ± 0.2)°, (10.6 ± 0.2)°, (12.8 ± 0.2)°, (14.4 ± 0.2)°, (17.5 ± 0.2)°, (22.9 ± 0.2)°, (23.5 ± 0.2)° and (25.0 ± 0.2)°; or

[0265] (10.0 ± 0.2)°, (10.6 ± 0.2)°, (12.8 ± 0.2)°, (14.4 ± 0.2)°, (17.5 ± 0.2)°, (18.1 ± 0.2)°, (22.9 ± 0.2)°, (23.5 ± 0.2)° and (25.0 ± 0.2)°; or

[0266] (10.0 ± 0.2)°, (10.6 ± 0.2)°, (11.1 ± 0.2)°, (12.8 ± 0.2)°, (14.4 ± 0.2)°, (17.5 ± 0.2)°, (18.1 ± 0.2)°, (22.9 ± 0.2)°, (23.5 ± 0.2)° and (25.0 ± 0.2)°; or

[0267] (10.0 ± 0.2)°, (10.6 ± 0.2)°, (11.1 ± 0.2)°, (12.8 ± 0.2)°, (14.4 ± 0.2)°, (17.5 ± 0.2)°, (18.1 ± 0.2)°, (20.0 ± 0.2)°, (22.9 ± 0.2)°, (23.5 ± 0.2)° and (25.0 ± 0.2)°; (when measured with Cu-Kα 1,2 radiation having a wavelength of 0.15419 nm at a temperature in the range of 20°C to 30°C)

[0268] In yet another embodiment, the present disclosure relates to a crystalline form of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (Hydrate A), which crystalline form is characterized by having an XRPD pattern substantially the same as that shown in 1,2 the present invention when measured at a temperature in the range of 20 °C to 30 °C with Cu-Kα Figure 4 radiation having a wavelength of 0.15418 nm.

[0269] In another embodiment, the present disclosure relates to a crystalline form of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (Hydrate A), which crystalline form is characterized by having a broad endothermic event with a peak maximum temperature of 109 ± 10 °C when measured by DSC at a heating rate of 10 K / min.

[0270] Hydrate B

[0271] In a further aspect, the present disclosure provides Hydrate B of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. Table 5 shows the most prominent X-ray powder diffraction peaks of crystalline Hydrate B of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate.

[0272] Table 5. Most prominent X-ray powder diffraction peaks of Hydrate B

[0273]

[0274] In one embodiment, the present disclosure provides Hydrate B of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, which Hydrate B is characterized by having an XRPD pattern comprising reflections at the following 2-theta angles when measured at a temperature in the range of 20 °C to 30 °C with Cu-Kα 1,2 radiation having a wavelength of 0.15419 nm:

[0275] (5.5 ± 0.2)°, (8.4 ± 0.2)° and (13.0 ± 0.2)°; or

[0276] (5.5 ± 0.2)°, (8.4 ± 0.2)°, (13.0 ± 0.2)° and (19.6 ± 0.2)°; or

[0277] (5.5 ± 0.2)°, (8.4 ± 0.2)°, (8.7 ± 0.2)°, (13.0 ± 0.2)° and (19.6 ± 0.2)°; or

[0278] (5.5 ± 0.2)°, (8.4 ± 0.2)°, (8.7 ± 0.2)°, (13.0 ± 0.2)°, (16.5 ± 0.2)° and (19.6 ± 0.2)°; or

[0279] (5.5 ± 0.2)°, (8.4 ± 0.2)°, (8.7 ± 0.2)°, (13.0 ± 0.2)°, (16.5 ± 0.2)°, (19.6 ± 0.2)° and (20.6 ± 0.2)°; or

[0280] (5.5 ± 0.2)°, (8.4 ± 0.2)°, (8.7 ± 0.2)°, (11.0 ± 0.2)°, (13.0 ± 0.2)°, (16.5 ± 0.2)°, (19.6 ± 0.2)° and (20.6 ± 0.2)°; or

[0281] (5.5 ± 0.2)°, (8.4 ± 0.2)°, (8.7 ± 0.2)°, (11.0 ± 0.2)°, (13.0 ± 0.2)°, (16.5 ± 0.2)°, (17.9 ± 0.2)°, (19.6 ± 0.2)° and (20.6 ± 0.2)°; or

[0282] (5.5 ± 0.2)°, (8.4 ± 0.2)°, (8.7 ± 0.2)°, (11.0 ± 0.2)°, (13.0 ± 0.2)°, (16.5 ± 0.2)°, (17.9 ± 0.2)°, (19.6 ± 0.2)°, (20.6 ± 0.2)° and (24.0 ± 0.2)°;

[0283] In yet another embodiment, the present disclosure relates to a crystalline form of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (hydrate B), which crystalline form is characterized by having an XRPD pattern substantially the same as that shown in 1,2 the present invention when measured with Cu-Kα Figure 5 radiation having a wavelength of 0.15418 nm at a temperature in the range of 20 °C to 30 °C.

[0284] In another embodiment, the present disclosure relates to a crystalline form of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (hydrate B), which is characterized by a broad endothermic event with a peak maximum temperature of 93 ± 10 °C when measured by DSC at a heating rate of 10 K / min.

[0285] In another aspect, the present invention relates to a composition comprising a crystalline form of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate as defined in any of the above embodiments, said composition being substantially free of any other solid forms of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. For example, a composition of the present disclosure comprising the crystalline hydrate A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate comprises, based on the weight of the composition, at most 20 w-%, preferably at most 10 w-%, more preferably at most 5 w-%, 4 w-%, 3 w-%, 2 w-% or 1 w-% of any other solid forms of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate.

[0286] Ca V 1.2-HEK293(AUX) cell line

[0287] In one aspect, the present disclosure relates to cells that comprise a human CaV1.2 α1C (alpha1C) subunit (encoded by human CACNA1C transcript variant 14; GenBank ID: NM_001129840), an α2Δ (alpha2delta, α2Δ2) auxiliary subunit (encoded by human CACNA2D2 transcript variant 3; GenBank ID: NM_001174051), and a β2 (beta2) auxiliary subunit (encoded by human CACNB2 transcript variant 2; GenBank ID: NM_201596), wherein the α1C subunit is constitutively expressed, and the α2Δ2 subunit and the β2 subunit are doxycycline-inducible. In another aspect, the present disclosure relates to cells for use in a method of screening for biologically active compounds. In one aspect, the cells are contained in a stable cell line (e.g., HEK293) that constitutively expresses the α1C subunit and doxycycline-inducibly expresses the α2Δ2 subunit and the β2 subunit. In another aspect, the stable cell line is generated by DNA transfection or viral transduction, e.g., via lentivirus or baculovirus. In one aspect, the cells are contained in a cell line from transient DNA transfection (e.g., HEK293) that constitutively expresses the α1C subunit and doxycycline-inducibly expresses the α2Δ2 subunit and the β2 subunit. In another aspect of the present disclosure, the compound is a Ca V 1.2 activator. In another aspect, the present disclosure relates to cells for use in a method of identifying an agent for treating, preventing, and / or diagnosing a disease or disorder (e.g., schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome). In some aspects, the agent is a Ca V 1.2 activator.

[0288] Intermediates and Examples

[0289] The following examples are intended to illustrate the present disclosure and should not be construed as limiting thereof.

[0290] These examples were separated into their single enantiomers and tested in the Sophion Qpatch assay described in the Biological Data section below. However, the stereochemistry of each enantiomer has not been determined. The stereochemistry of the active enantiomers of Example 6 (methyl (R)-4-(2-((S)-1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate) and Example 9 (methyl (R)-4-(2-(difluoromethoxy)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate) was analyzed using single crystal x-ray crystallography, and it was determined that the active enantiomer was the R-enantiomer. Accordingly, it is assumed that the R-enantiomers of the methyl 2-(fluoromethyl)-5-oxo-4-phenyl-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate and methyl 2-(fluoromethyl)-5-oxo-4-phenyl-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate compounds depicted below are the active enantiomers.

[0291]

[0292] Methyl (R)-2-(fluoromethyl)-5-oxo-4-phenyl-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[0293]

[0294] Methyl (R)-2-(fluoromethyl)-5-oxo-4-phenyl-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0295] The example numbers of all active enantiomers assumed to have the R configuration are given (Examples 1, 2, 3, etc.). All other isomers isolated from the synthesis are given example numbers with letters (Examples 1b, 2b, 3b, etc.). Although there is strong evidence that the R configuration is the desired stereochemistry, it is still possible that the S-enantiomer is the active enantiomer.

[0296] Temperatures are given in degrees Celsius. Unless otherwise stated, all evaporations are carried out under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (= 20 - 133 mbar). The structures of the end products, intermediates, and starting materials were confirmed by standard analytical methods (e.g., microanalysis and spectroscopic characteristics (e.g., MS, IR, NMR)).

[0297] All starting materials, structural units, reagents, acids, bases, dehydrating agents, solvents, and catalysts used to synthesize the compounds of the present disclosure are commercially available or can be prepared by organic synthesis methods known to those of ordinary skill in the art. In addition, the compounds of the present disclosure can be prepared by organic synthesis methods known to those of ordinary skill in the art, as shown in the following examples. The abbreviations used are conventional abbreviations in the art or the following abbreviations:

[0298] 1 H NMR Proton nuclear magnetic resonance

[0299] AUX Ca V 1.2-channel auxiliary subunit

[0300] C Celsius

[0301] CD3OD Methanol-d4

[0302] CDCl3 Chloroform-d CHO Chinese hamster ovary cells

[0303] Ct Threshold cycle in quantitative polymerase chain reaction assay

[0304] d Doublet

[0305] DAST Diethylaminosulfur trifluoride

[0306] DCM Dichloromethane

[0307] dd Double doublet

[0308] DME 1,4-Dimethoxyethane

[0309] DMEM Dulbecco's Modified Eagle Medium

[0310] DMF N,N-Dimethylformamide

[0311] DMSO Dimethyl sulfoxide

[0312] DMSO-d6 Dimethyl sulfoxide-d6

[0313] D-PBS Dulbecco's Phosphate Buffered Saline

[0314] EC50 Half maximal effective concentration

[0315] EDTA Ethylenediaminetetraacetic acid

[0316] EGTA Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

[0317] Eq Equivalent

[0318] EtOAc Ethyl acetate

[0319] FAM 6-Carboxyfluorescein

[0320] FCS Furin cleavage site

[0321] FRT Flp recombinase recognition target site

[0322] g gram

[0323] h hour

[0324] H2O Water

[0325] HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

[0326] HOBt 1-Hydroxy-7-azabenzotriazole

[0327] HPLC High Performance Liquid Chromatography

[0328] HRMS High Resolution Mass Spectrometry

[0329] Hrs Hours

[0330] Hz Hertz

[0331] IACUC Institutional Animal Care and Use Committee

[0332] IPA Isopropyl alcohol

[0333] kg kilogram

[0334] L liter

[0335] LCMS Liquid Chromatography-Mass Spectrometry

[0336] M mole

[0337] m multiplet

[0338] m / z Mass-to-charge ratio

[0339] mg milligram

[0340] MHz Megahertz

[0341] min minute

[0342] mL milliliter

[0343] ml milliliter

[0344] mL / min Milliliter per minute

[0345] mm millimeter

[0346] mM millimole

[0347] mmol millimole

[0348] mRNA Messenger Ribonucleic Acid

[0349] MS Mass Spectrometry

[0350] mV Millivolt μl

[0351] N Normal Concentration

[0352] n-BuLi n-Butyllithium

[0353] NMR Nuclear Magnetic Resonance

[0354] NOESY Nuclear Overhauser Effect Spectroscopy

[0355] pCMV Cytomegalovirus Promoter

[0356] P2A Peptide Self-Cleaving Sequence Derived from Porcine Teschovirus-1

[0357] PdCl2(dppf) Dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II)

[0358] PD Pharmacodynamics

[0359] PK Pharmacokinetics

[0360] ppm Parts Per Million

[0361] PyBOP Benzotriazol-1-yloxytris(pyrrolidino)phosphonium Hexafluorophosphate

[0362] QT Time Interval between Q Wave and T Wave of Electrocardiogram

[0363] rac Racemic

[0364] rpm Revolutions Per Minute

[0365] RNA Ribonucleic Acid

[0366] RT Room Temperature

[0367] Rt Retention Time

[0368] RT-PCR Reverse Transcription-Polymerase Chain Reaction

[0369] s Singlet

[0370] SFC Supercritical Fluid Chromatography

[0371] SFM Serum-Free Medium

[0372] SNP Single Nucleotide Polymorphism

[0373] t Triplet

[0374] TFA Trifluoroacetic acid

[0375] THF Tetrahydrofuran

[0376] uL Microliter

[0377] Um Micrometer

[0378] UPLC Ultra Performance Liquid Chromatography

[0379] UV Ultraviolet

[0380] VIC 2′-Chloro-7′-phenyl-1,4-dichloro-6-carboxy-fluorescein

[0381] v / v Volume / volume percentage

[0382] Small molecule X-ray crystallography

[0383] Data collection

[0384] Intensity data were collected at 100 K on a Bruker AXS three-circle diffractometer with monochromatic Cu(Kα)-radiation, a microfocus rotating anode generator, and a Smart 6000 CCD detector using SMART software (Bruker AXS (2003)). Sixteen ω scans were performed at different Φ-positions to ensure adequate data redundancy. Data processing and global unit cell refinement were carried out using Saint (Bruker AXS (2012)). A semi-empirical absorption correction was applied, which was based on the intensities of symmetry-related reflections measured at different angular settings using SADABS 2016 / 2 version (Krause L (2015)). The extinction coefficient was refined to 0.00048(11). Crystal data, data collection parameters, and refinement results are listed.

[0385] Structure solution and refinement

[0386] The structure was solved by the dual-space recycling method and subsequent DF synthesis and refined based on full-matrix least-squares on F2 using the SHELXTL program suite (Sheldrick GM (2001)) with SHELXL-2013 / 4.

[0387] References:

[0388] Allen FH, Kennard O, Watson D et al (1987) Tables of Bond Lengths determined by X-Ray and Neutron Diffraction. Part 1, Bond Lengths in Organic Compounds. J. Chem. Soc. Perkin Trans II; S1 - S19.

[0389] Bruker AXS (2005) SMART V5.632. Bruker AXS Inc., Madison, WI, USA.

[0390] Bruker AXS (2012) SAINT V7.36A. Bruker AXS Inc., Madison, WI, USA.

[0391] Krause L, Herbst-Irmer R, Sheldrick GM et al (2015) Comparison of silver and molybdenum microfocus X-ray sources for single-crystal structure determination. J. Appl. Cryst.; 48:3 - 10.

[0392] Spek AL (2003) Single-crystal structure validation with the program PLATON. J. Appl.

[0393] Cryst.; 36:7 - 13.

[0394] Sheldrick GM (2001) SHELXTL V6.12. Bruker AXS Inc.. Madison, WI, USA.

[0395] LCMS Method 1:

[0396] Instrument: Waters Acquity UPLC, photodiode array detector; Column: Acquity UPLC BEH C 181.7 μm, 21 x 30 mm; 2 min runtime, 2% solvent B from 0 to 0.1 min, 2% → 98% solvent B: solvent A from 0.1 to 1.8 min, 98% solvent B for 0.2 min. Solvents: solvent A = 0.1% formic acid (v / v) in water, solvent B = 0.1% formic acid (v / v) in acetonitrile. Injection volume 2 - 5 μL; UV detection array 210 - 400, mass detection 120 - 1250 (electrospray ionization); column, at 50 °C; flow rate 1.0 mL / min.

[0397] LCMS Method 2:

[0398] Instrument: Waters Acquity UPLC, photodiode array detector; column: Acquity UPLC BEH C 18 1.7 μm 21 x 50 mm; 2 min runtime, 2% solvent B from 0 to 0.1 min, 2% → 98% solvent B: solvent A from 0.1 to 1.8 min, 98% solvent B for 0.2 min. Solvents: solvent A = 5 mM ammonium hydroxide in water, solvent B = 5 mM ammonium hydroxide in acetonitrile. Injection volume 2 - 5 μL; UV detection array 210 - 400, mass detection 120 - 1250 (electrospray ionization); column, at 50 °C; flow rate 1.0 mL / min.

[0399] LCMS Method 3:

[0400] Instrument: Waters Acquity UPLC, photodiode array detector; column Acquity UPLC BEH C 18 1.7 μm 21 x 30 mm; 5.2 min runtime, 2% → 98% solvent B: solvent A from 0 to 5.15 min, 98% solvent B from 5.15 to 5.20 min. Solvents: solvent A = 0.1% formic acid (v / v) in water, solvent B = 0.1% formic acid (v / v) in acetonitrile. Injection volume 2 - 5 μL; UV detection array 210 - 400, mass detection 120 - 1600; column, at 50 °C, flow rate 1.0 mL / min.

[0401] LCMS Method 4:

[0402] Instrument: Waters Acquity UPLC, photodiode array detector; column Acquity UPLC BEH C 181.7 μm, 21 x 30 mm; 5.2 min runtime, 2% → 98% solvent B: solvent A from 0 to 5.15 min, 98% solvent B from 5.15 to 5.20 min. Solvents: solvent A = 5 mM ammonium hydroxide in water, solvent B = 5 mM ammonium hydroxide in acetonitrile. Injection volume 2 - 5 μL; UV detection array 210 - 400, mass detection 120 - 1600; column, at 50 °C, flow rate 1.0 mL / min.

[0403] LCMS Method 5:

[0404] Instrument: Agilent 1200 LC / G1956A, diode array detector; column: Chromolith Flash C 18 , 1.6 μm, 2 x 25 mm; 1.5 min runtime, 5% → 95% solvent B: solvent A (from 0 → 1.2 min) and then 95% solvent B (from 1.21 → 1.5 min). Solvents: solvent A = 0.0375% TFA (v / v) in water, solvent B = 0.01875% TFA (v / v) in acetonitrile. Injection volume 2 - 5 μL; UV detection 220 and 254 nM, mass detection 100 - 1000 (electrospray ionization); column, at 50 °C; flow rate 1.5 mL / min.

[0405] LCMS Method 6:

[0406] Instrument: SHIMADZU LCMS - 2020, photodiode array detector; column: Kinetex EVO C 18 , 5 uM, 1 x 30 mm; 1.55 min runtime, 5% → 95% solvent B: solvent A (from 0 → 1.20 min) and then 95% solvent B (from 1.21 min to 1.55 min). Solvents: solvent A = 0.025% NH4OH (v / v) in water, solvent B = acetonitrile. Injection volume 2 - 5 μL; UV detection 220 and 254 nM, mass detection 100 - 1000 (electrospray ionization); column, at 40 °C; flow rate 1.5 mL / min.

[0407] LCMS Method 7:

[0408] Instrument: API2000, column: Mercury MS Synergi 2μm, 20X4.0 mm, C12; gradient: A - 0.1% formic acid in water / B - acetonitrile: time / %B: 0.0 / 30, 0.5 / 30, 1.5 / 95, 2.0 / 95, 2.5 / 30, 3.0 / 30, flow rate 2.0 mL / min; UV detection array 190 - 400, mass detection 100 - 1000 (electrospray ionization); column temperature 30°C.

[0409] LCMS Method 8:

[0410] Instrument: API3000, column: Synergi 2.5μm MAX-RP, 20X4.0 mm 100A Mercury; gradient: 0.1% formic acid in water, B: acetonitrile: time / %B 0 / 10, 0.5 / 20, 1.5 / 95, 2.0 / 95, 2.5 / 10, 3 / 10, flow rate 2.0 mL / min; UV detection array 190 - 400 (total wavelength chromatogram), mass detection 100 - 1000 (electrospray ionization); column temperature 30°C.

[0411] LCMS Method 9:

[0412] Instrument: API3000, column: Synergi 2.5μm, 50x4.6 mm, MAX-RP 100A; gradient: 0.1% formic acid in water, B: acetonitrile: time 0.0 / 10, 0.2 / 50, 1.0 / 95, 1.5 / 100, 2.5 / 95, 2.9 / 50, 3.2 / 10, 4 / 10, flow rate 1.2 mL / min; UV detection array 190 - 400 (total wavelength chromatogram), mass detection 100 - 1000 (electrospray ionization); column temperature 30°C.

[0413] LCMS Method 10:

[0414] Shimadzu, column: Mercury MS Synergi 2.5μm, 20X4.0 mm, C12; gradient: A - 0.1% formic acid in water / B - acetonitrile: time / %B: 0.1 / 5, 0.5 / 5, 1.0 / 95, 1.5 / 95, 2.0 / 5, 3.0 / 5, flow rate 2.0 mL / min; UV detection array 200 - 400, mass detection 100 - 1000 (electrospray ionization); column temperature 40°C.

[0415] LCMS Method 11:

[0416] Shimadzu, column: Kinetex 5μm EVO C18 100A, (100x2.1 mm); gradient: A - 0.1% formic acid in water / B - acetonitrile: time / %B: 0 / 5, 1 / 30, 3 / 95, 4 / 95, 4.1 / 5, 6 / 5, flow rate 1.4 mL / min; UV detection array 200 - 400, mass detection 100 - 1000 (electrospray ionization); column temperature 40 °C.

[0417] LCMS Method 12:

[0418] Shimadzu, column: Synergi 2.5μm MAX-RP 100A, (20X4.0 mm) Mercury; gradient: A - 0.1% formic acid in water / B - acetonitrile: time / %B: 0.1 / 5, 0.5 / 5, 1.0 / 95, 1.5 / 95, 2.0 / 5, 3.0 / 5, flow rate 2.0 mL / min; UV detection array 200 - 400, mass detection 100 - 1000 (electrospray ionization); column temperature 40 °C.

[0419] LCMS Method 13:

[0420] Acquity, column: UPLC BEH C18 1.7μm 2.1x50 mm; gradient: A - 0.1% formic acid in water / B - acetonitrile: 2.2 min run time, 2% → 98% solvent B: solvent A (from 0 → 1.76 minutes) and then 95% solvent B (from 1.76 minutes to 2.0 minutes); flow rate 1.0 mL / min; UV detection array 210 - 400 nm; mass range 100 - 2050 Da; HRMS_2 min; column temperature 50 °C for preparative HPLC method for purification:

[0421] Method 1: HPLC column: XBRIDGE-C18 (19.0x 150mm, 5 microns), mobile phase - A: 0.1% TFA in H2O, B: CH3CN, gradient (time / %B): 0 / 20, 2 / 20, 8 / 50) flow rate: [19 mL / min].

[0422] Method 2: HPLC column: ZORBAX ECLIPSE XDB C18 (21.2X 150mm, 5 microns), mobile phase - A: 0.1% TFA in H2O, B: CH3CN, gradient (time / %B): 0 / 10, 2 / 20, 10 / 40, flow rate: [20 mL / min].

[0423] Method 3: HPLC [Column: XBRIDGE C18 (21.2x 150mm, 5 µm), Mobile phase - A: 10 mM NH4HCO3 in water, B: CH3CN, Gradient (time / %B): 0 / 10, 2 / 20, 8 / 50, Flow rate: [18 mL / min].

[0424] Method 4: HPLC Column: Gemini NX C18 (21.2x 150.00mm, 5 µm); Mobile phase - (A): 0.1% TFA in water (B): Acetonitrile / Methanol; Flow rate: 15 mL / min; (time / %B 0 / 20, 2 / 20, 8 / 20)

[0425] Method 5: HPLC Column: KINETEX EVO 5μ C18 (21.2X 150mm), Mobile phase: water (A) CH3CN (B), Gradient (time / %B): 0 / 20, 2 / 30, 7 / 70, and Flow rate: [18 mL / min].

[0426] Method 6: HPLC Column: KINETEX C18, (21.2x 150mm), Mobile phase: A: water, B: CH3CN:MeOH, Gradient (time / %B): 0 / 20, 2 / 30, 7 / 70, Flow rate: 18 mL / min].

[0427] Method 7: HPLC Column: KINETEX (21.2x 150mm, 5 µm), Mobile phase: A = 0.05% TFA in water, B = CH3CN, Gradient (time / %B): 0 / 20, 2 / 30, 10 / 60, Flow rate: 20 mL / min].

[0428] Chiral preparative HPLC methods for separating isomers:

[0429] Method 1: Column: CHIRALPAK IC (10x 250mm, 5 µm), Mobile phase: hexane (A) IPA:MeOH, 1:1 (B); Flow rate: 8 mL / min; Isocratic: 96:04 (A:B).

[0430] Method 2: Column: REGIS WELKO (250x 10mm, 5 µm), Mobile phase: hexane (A):EtOH, 1:1 (B); Flow rate: 9 mL / min; Isocratic: 85:15 (A:B).

[0431] Method 3: Column: CHIRALPAC IG (250x 10mm, 5 µm), Mobile phase: IPA (A):MeOH, 1:1 (B); Flow rate: 6 mL / min; Isocratic: 98:2 (A:B).

[0432] Method 4: Column: LUX cellulose-4 (10 x 250 mm, 5 µm), mobile phase: hexane (A), EtOH:IPA 1:1 (B); flow rate: 8 mL / min; isocratic: 90:10 (A:B).

[0433] Method 5: Column: LUX cellulose-4 (10 x 250 mm, 5 µm), mobile phase: hexane (A), EtOH: 1:1 (B); flow rate: 8 mL / min; isocratic: 90:10 (A:B).

[0434] Method 6: Column: LUX cellulose-4 (10 x 250 mm, 5 µm), mobile phase: hexane (A); EtOH:MeOH 1:1 (B); flow rate: 18 mL / min; isocratic: 90:10 (A:B).

[0435] Method 7: Column: LUX cellulose-4 (21.2 x 250 mm, 5 µm), mobile phase: hexane (A); EtOH:MeOH 1:1 (B); flow rate: 19 mL / min; isocratic: 80:20 (A:B).

[0436] Method 8: Column: REGIS (10X 250 mm, 5 µm), mobile phase: CO2 (A), MeOH:EtOH 1:1 (B); flow rate: 13 mL / min, isocratic: 80:20 (A:B).

[0437] Method 9: Column: LUX cellulose-4 (10 x 250 mm, 5 µm), mobile phase: hexane (A) 0.1% DEA in EtOH:MeOH (50:50) (B); flow rate: 7 mL / min; isocratic: 93:07 (A:B).

[0438] Chiral analytical HPLC methods for the analysis and separation of isomers:

[0439] Method 1: Column: Lux, cellulose-4 (250X 4.6 mm, 5 µm); mobile phase: A = n-hexane, B = 0.1% TFA in ethanol; 1 mL / min; isocratic: 70:30 (A:B)

[0440] Method 2: Column: LUX cellulose-4 (4.6 x 250 mm, 5 µm); mobile phase: hexane (A), EtOH: 1:1 (B); flow rate: 1 mL / min; isocratic: 50:50 (A:B).

[0441] Method 3: Column: REGIS, (S,S)WHELK-01 (250X 4.6 mm, 5 μm); Mobile phase: A = n-hexane, B = ethanol; Flow rate: 1 mL / min; Isocratic: 70:30 (A:B).

[0442] Intermediate A: Formation of ethyl 4-acetoxy-3-oxobutyrate

[0443]

[0444] To a solution of ethyl 4-chloro-3-oxobutyrate (200 g, 1215.1 mmol) in acetic acid (1500 mL) was added potassium acetate (357 g, 3645.4 mmol). The resulting solution was stirred at 90 °C for 18 h. The solvent was dissolved in water (2 L) and extracted into ethyl acetate (1 L x 4 times). The EtOAc phases were combined, washed with saturated NaHCO3 solution (2 L), brine, and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (0→15% ethyl acetate in petroleum ether) to afford the title compound ethyl 4-acetoxy-3-oxobutyrate (152 g) as a pale brown liquid.

[0445] 1 H NMR (400 MHz, CDCl3) δ 4.78 (s, 2H), 4.20 (q, J = 14.1, 7.2 Hz, 2H), 3.49 (s, 2H), 2.16 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H).

[0446] Intermediate B: Formation of methyl (Z)-3-amino-4-fluorobut-2-enoate

[0447] Method 1:

[0448]

[0449] Under a nitrogen atmosphere, a solution of methyl acetate (36.5 g, 492.71 mmol) in tetrahydrofuran (200 mL) was cooled to -78 °C. Lithium diisopropylamide (246.44 mL, 2.0 M, 492.71 mmol) in THF was slowly added to the reaction over a 20 min period. The resulting mixture was stirred at -78 °C for 1 h, and then 2-fluoroacetonitrile (19.4 g, 328.9 mmol) in tetrahydrofuran (150 mL) solution was added dropwise. The reaction mixture was stirred at -78 °C for an additional 1 h, then saturated ammonium chloride solution (200 mL) was added, and the product was extracted into ethyl acetate (5 L). The EtOAc was washed with saturated brine solution (500 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to afford the title compound methyl (Z)-3-amino-4-fluorobut-2-enoate as a white crystalline solid (16 g).

[0450] Method 2:

[0451]

[0452] Step 1: Under a nitrogen atmosphere, a solution of methyl acetate (83.78 g, 1131.0 mmol) in tetrahydrofuran (800 mL) was cooled to -78 °C, and then lithium diisopropylamide (565.5 mL, 2.0 M, 1131.0 mmol) in THF was slowly added to the reaction over a 20 min period. The resulting mixture was stirred at -78 °C for 1 h, then ethyl 2-fluoroacetate (100 g, 942.5 mmol) in tetrahydrofuran (200 mL) solution was added dropwise, and the reaction mixture was stirred at -78 °C for an additional 1 h. Saturated ammonium chloride solution (200 mL) was added to the reaction mixture, and the product was extracted into ethyl acetate (5 L). The EtOAc was washed with saturated brine solution (500 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to give yellow crystals of methyl 4-fluoro-3-oxobutanoate (60 g).

[0453] 1 1H NMR (400 MHz, CDCl3) δ 4.97 (s, 1H), 4.85 (s, 1H), 3.76 (d, J = 2.3 Hz, 3H), 3.62 (d, J = 3.7 Hz, 2H).

[0454] Step 2: At room temperature in a sealed tube, saturated ammonia solution in methanol (300 mL) was added to methyl 4-fluoro-3-oxobutanoate (from Step 1, 60 g). The resulting mixture was stirred at room temperature for 16 h. The solvent was removed under reduced pressure to give the title compound methyl (Z)-3-amino-4-fluorobut-2-enoate as a white solid (51 g).

[0455] 1 H NMR (300 MHz, DMSO-d6) δ 4.98 (t, J = 0.7, 0.7 Hz, 1H), 4.82 (t, J = 0.7, 0.7 Hz, 1H), 4.53 (q,, 1H), 3.53 (d, J = 1.2 Hz, 3H).

[0456] General Procedure I

[0457]

[0458] Step 1: To a solution of aldehyde (1 mmol) in EtOH (10 mL) was added methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 1 mmol or 1.2 mmol) and ethyl 4-acetoxy-3-oxobutanoate (Intermediate A, 1 mmol or 1.2 mmol). The resulting solution was stirred at 80 °C for 16 h. The solvent was removed under reduced pressure to give the title compound (crude).

[0459] Step 2: Potassium carbonate (5 mmol) was added to the crude intermediate from Step 1 in methanol (10 mL). The resulting solution was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and it was added to water. The product was extracted into ethyl acetate (100 mL), washed with brine, and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel.

[0460] General Procedure II

[0461]

[0462] To a solution of aldehyde (1 mmol) in MeOH (10 mL) was added methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 1 mmol or 1.2 mmol) and ethyl 4-acetoxy-3-oxobutanoate (Intermediate A, 1 mmol or 1.2 mmol). The resulting solution was stirred at 75 °C for 16 - 48 h. The solvent was removed under reduced pressure and the product was purified using flash chromatography on silica gel.

[0463] Example 1: Methyl (R)-4-(2-((R or S)-2,2-difluorocyclopropyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0464]

[0465] Step 1: 2-(2-Bromo-3-fluorophenyl)-1,3-dioxolane

[0466]

[0467] To a solution of 2-bromo-3-fluorobenzaldehyde (60 g, 295.56 mmol) and ethylene glycol (65.4 mL, 1182.2 mmol) in toluene (600 mL) was added p-toluenesulfonic acid monohydrate (28.11 g, 147.78 mmol). The resulting solution was stirred at 120 °C for 24 h using a dean-stark apparatus. The reaction mixture was poured into water (2 L) and extracted into ethyl acetate (3 L). The EtOAc was washed with saturated NaHCO3 solution (1 L), brine, and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to afford the title compound 2-(2-bromo-3-fluorophenyl)-1,3-dioxolane (60 g) as a colorless liquid.

[0468] 1 1H NMR (400 MHz, CDCl3) δ 7.42 - 7.37 (m, 1H), 7.34 - 7.29 (m, 1H), 7.16 - 7.10 (m, 1H), 6.10 (s, 1H), 4.19 - 4.09 (m, 4H).

[0469] Step 2: 2-(3-Fluoro-2-vinylphenyl)-1,3-dioxolane

[0470]

[0471] To a solution of 2-(2-bromo-3-fluorophenyl)-1,3-dioxolane (from Step 1, 15 g, 60.72 mmol) and potassium vinyltrifluoroborate (16.26 g, 121.45 mmol) in isopropanol (200 mL) was added triethylamine (25.5 mL, 182.16 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).DCM (4.95 g, 6.07 mmol). The resulting solution was degassed with argon for 10 min and stirred at 80 °C for 16 h. The reaction mixture was filtered and washed with ethyl acetate (100 mL). The filtrate was added to water (1 L) and the product was extracted into ethyl acetate (2 L). The EtOAc phase was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to give the title compound 2-(3-fluoro-2-vinylphenyl)-1,3-dioxolane (7.5 g) as a colorless liquid.

[0472] 1 H NMR (400 MHz, CDCl3) δ 7.45 - 7.41 (m, 1H), 7.27 - 7.20 (m, 1H), 7.10 - 7.03 (m, 1H), 6.82 (dd, J = 17.7, 11.7 Hz, 1H), 5.82 - 5.78 (m, 1H), 5.66 - 5.60 (m, 1H), 5.95 (s, 1H), 4.18 - 4.02 (m, 4H).

[0473] Step 3: 2-(2-(2,2-difluorocyclopropyl)-3-fluorophenyl)-1,3-dioxolane

[0474]

[0475] To a solution of 2-(3-fluoro-2-vinylphenyl)-1,3-dioxolane (from Step 2, 6.5 g, 33.47 mmol) and trimethyl(trifluoromethyl)silane (50 mL, 334.7 mmol) in tetrahydrofuran (120 mL) was added sodium iodide (2.5 g, 16.73 mmol). The resulting solution was stirred at 65 °C for 24 h. The solvent was evaporated under reduced pressure and the residue was dissolved in dichloromethane (100 mL). This was washed with water (50 mL), brine and dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound 2-(2-(2,2-difluorocyclopropyl)-3-fluorophenyl)-1,3-dioxolane (8 g) as a colorless liquid.

[0476] 11H NMR (400 MHz, CDCl3) δ 7.42 (d, J = 7.5 Hz, 1H), 7.34 - 7.28 (m, 1H), 7.16 - 7.03 (m, 1H), 6.06 (s, 1H), 4.20 - 4.01 (m, 4H), 2.77 - 2.66 (m, 1H), 2.02 - 1.72 (m, 2H).

[0477] Step 4: 2-(2,2-difluorocyclopropyl)-3-fluorobenzaldehyde

[0478]

[0479] To a solution of 2-(2-(2,2-difluorocyclopropyl)-3-fluorophenyl)-1,3-dioxolane (from Step 3, 8 g, 32.76 mmol) in diethyl ether (80 mL) was added 6N HCl (10 mL). The resulting solution was stirred at room temperature for 2 h. This was washed with water (50 mL), saturated NaHCO3 solution (100 mL), brine, and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (0→5% ethyl acetate in petroleum ether) to afford the title compound 2-(2,2-difluorocyclopropyl)-3-fluorobenzaldehyde (3.8 g) as a colorless liquid.

[0480] 1 1H NMR (400 MHz, CDCl3) δ 10.28 (s, 1H), 7.70 (d, J = 6.9 Hz, 1H), 7.50 - 7.43 (m, 1H), 7.36 - 7.26 (m, 1H), 2.92 - 2.81 (m, 1H), 2.16 - 2.06 (m, 1H), 1.67 - 1.53 (m, 1H).

[0481] Step 5: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(2,2-difluorocyclopropyl)-3-fluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0482]

[0483] Step 1 of General Procedure I was used to synthesize the title compound (using the aldehyde from Step 4 (1.0 g, 4.996 mmol), Intermediate A (945 mg, 4.996 mmol), and Intermediate B (665 mg, 4.996 mmol)) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(2,2-difluorocyclopropyl)-3-fluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (1.4 g, crude). The crude product was used as such in the next step without further purification and analysis.

[0484] Step 6: Methyl 4-(2-(2,2-difluorocyclopropyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0485]

[0486] Step 2 of General Procedure I was used to synthesize the title compound (using the intermediate from Step 5, 1.4 g, 2.884 mmol). The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to give the title compound methyl 4-(2-(2,2-difluorocyclopropyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate as an off-white solid (125 mg). The sample was chiral separated into two products using chiral SFC: column: Lux-cellulose-2 21x250 mm 5um; flow rate: 80 g / min; co-solvent: 20% 1:1 MeOH:IPA; detection: 344 nm; BPR set point: 125 bar.

[0487] The previously separated Peaks 1 and 2 were separated into 4 isomers using chiral SFC: column: WO1 SS 21x 250mm; flow rate: 80 g / min; co-solvent: 15% IPA; detection: 344 nm; BPR set point: 150 bar.

[0488] Example 1

[0489] 20 mg of the title compound as the second eluting stereoisomer as a white solid (61%)

[0490] SFC Rt = 2.49; mobile phase: 5%-55% (1:1) MeOH IPA w / 10 mM NH4OH in CO2, 5 mL / min; column: Lux cellulose-2 4.6x 100mm 5μm

[0491] LCMS Rt = 2.12 min; MS m / z 398.3 [M+H]+; [Method 3]

[0492] 1 1H NMR (400 MHz, DMSO-d6) δ 10.13 (d, J = 3.3 Hz, 1H), 7.31 (td, J = 8.0, 5.6 Hz, 1H), 7.20 - 6.83 (m, 2H), 5.78 - 5.41 (m, 2H), 5.22 (s, 1H), 5.03 - 4.73 (m, 2H), 3.51 (s, 3H), 2.86 (td, J = 12.4, 8.5 Hz, 1H), 2.64 - 2.53 (m, 1H), 2.18 (dt, J = 12.2, 5.5 Hz, 1H).

[0493] Example 1b

[0494] 15 mg of the first eluted stereoisomer as a white solid (46%)

[0495] SFC Rt = 2.42; Mobile phase: 5% - 55% (1:1) MeOH IPA w / 10 mM NH4OH in CO2, 5 mL / min; Column: Lux cellulose-2 4.6 x 100 mm 5 μm

[0496] LCMS Rt = 2.15 min; MS m / z 398.2 [M+H]+; [Method 3]

[0497] 1 1H NMR (400 MHz, DMSO-d6) δ 10.08 (d, J = 3.2 Hz, 1H), 7.33 (td, J = 8.1, 5.8 Hz, 1H), 7.25 - 6.92 (m, 2H), 5.94 - 5.54 (m, 2H), 5.27 (s, 1H), 4.79 (d, J = 1.4 Hz, 2H), 3.44 (s, 3H), 3.15 - 3.04 (m, 1H), 2.35 - 2.19 (m, 1H), 1.88 (dtd, J = 14.0, 8.2, 3.2 Hz, 1H).

[0498] Example 1c

[0499] 24 mg of the third eluted stereoisomer as a white solid (73%)

[0500] SFC Rt = 2.73 min; Mobile phase: 5% - 55% (1:1) MeOH IPA w / 10 mM NH4OH in CO2, 5 mL / min; Column: Lux cellulose-2 4.6 x 100 mm 5 μm

[0501] LCMS Rt = 2.16 min; MS m / z 398.3 [M+H]+; [Method 3]

[0502] 1 H NMR (400 MHz, DMSO-d6) δ 10.13 (d, J = 3.3 Hz, 1H), 7.31 (td, J = 8.0, 5.6 Hz, 1H), 7.18 - 6.87 (m, 2H), 5.61 (dd, J = 47.8, 4.0 Hz, 2H), 5.22 (s, 1H), 4.98 - 4.78 (m, 2H), 3.51 (s, 3H), 2.86 (td, J = 12.5, 8.5 Hz, 1H), 2.64 - 2.55 (m, 1H), 2.18 (tt, J = 12.8, 6.8 Hz, 1H).

[0503] Example 1d

[0504] 15 mg of the fourth eluted stereoisomer as a white solid (46%)

[0505] SFC Rt = 2.78 min; Mobile phase: 5% - 55% (1:1) MeOH IPA w / 10 mM NH4OH in CO2, 5 mL / min; Column: Lux cellulose-2 4.6 x 100 mm 5 μm

[0506] LCMS Rt = 2.11 min; MS m / z 398.1 [M+H]+; [Method 3]

[0507] 1 H NMR: (400 MHz, DMSO-d6) δ 10.08 (d, J = 3.3 Hz, 1H), 7.33 (td, J = 8.0, 5.6 Hz, 1H), 7.14 - 6.94 (m, 2H), 5.72 (dd, J = 47.8, 4.8 Hz, 2H), 5.27 (s, 1H), 4.79 (d, J = 1.4 Hz, 2H), 3.44 (s, 3H), 3.15 (d, J = 14.7 Hz, 1H), 2.33 - 2.20 (m, 1H), 1.99 - 1.79 (m, 1H).

[0508] Example 2: Methyl (R)-4-(2-cyclopropyl-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0509]

[0510] Step 1: 2-Cyclopropyl-3-fluorobenzaldehyde

[0511]

[0512] To a solution of 2-bromo-3-fluorobenzaldehyde (15 g, 73.88 mmol) and cyclopropylboronic acid (7.61 g, 88.66 mmol) in toluene (160 mL) was added 2N K2CO3 (25.5 mL, 182.16 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).DCM (6.03 g, 7.38 mmol). The resulting solution was degassed with argon for 10 min and stirred at 100 °C for 4 h. The reaction mixture was filtered and washed with ethyl acetate (200 mL). The filtrate was added to water (500 mL), and the product was extracted into ethyl acetate (2 L). The EtOAc phase was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→1% ethyl acetate in petroleum ether) to give the title compound 2-(3-fluoro-2-vinylphenyl)-1,3-dioxolane (11.2 g) as a colorless liquid.

[0513] 1 1H NMR (300 MHz, CDCl3) δ 10.70 (s, 1H), 7.63 (dd, J = 1.2, 7.8 Hz, 1H), 7.35 - 7.15 (m, 2H), 2.14 - 2.02 (m, 1H), 1.19 - 1.09 (m, 2H), 0.85 - 0.75 (m, 2H).

[0514] Step 2: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(2-cyclopropyl-3-fluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0515]

[0516] The title compound was synthesized using Step 1 of General Procedure I (using the aldehyde from Step 1, 4.0 g, 24.36 mmol) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-cyclopropyl-3-fluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (7.0 g, crude).

[0517] LCMS Rt = 1.806 min; MS m / z 450.3 [M+H]+; [Method 7]

[0518] Step 3: Methyl 4-(2-cyclopropyl-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0519]

[0520] The title compound was synthesized in Step 2 using General Procedure I (using the intermediate from Step 2, 7.0 g, 15.57 mmol). The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to give the title compound methyl 4-(2-cyclopropyl-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate as an off-white solid (1.15 g).

[0521] Using chiral preparative HPLC [Method 6], 150 mg of the racemic mixture was separated into its enantiomers.

[0522] Example 2

[0523] The first eluted enantiomer, 50 mg, obtained as a white solid.

[0524] Chiral HPLC Rt = 6.129 min [Chiral Analytical Method 1]

[0525] LCMS Rt = 1.538 min; MS m / z 360.0 [M-H]−; [Method 7]

[0526] 1 1H NMR (400 MHz, CDCl3) δ 7.15 - 7.05 (m, 1H), 6.92 (ddd, J = 7.8, 1.2, 0.6 Hz, 1H), 6.82 (ddd, J = 10.8, 8.2, 1.3 Hz, 1H), 5.78 (d, J = 0.9 Hz, 1H), 5.71 - 5.59 (m, 2H), 4.80 - 4.69 (m, 2H), 3.55 (s, 3H), 2.19 - 2.15 (m, 1H), 1.34 - 1.20 (m, 1H), 1.06 - 0.96 (m, 2H), 0.82 - 0.71 (m, 1H). No exchangeable NH was seen in the spectrum.

[0527] Example 3: Methyl (R)-4-(2-((R or S)-2,2-difluorocyclopropyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0528] And Example 4: Methyl (R)-4-(2-((R or S)-2,2-difluorocyclopropyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0529]

[0530] Step 1: 2-(2-Bromo-3,5-difluorophenyl)-1,3-dioxolane

[0531]

[0532] The title compound was synthesized according to a procedure similar to Step 1 of Example 1, using 10 g of 2-bromo-3,5-difluorobenzaldehyde to obtain 11.5 g of the desired product 2-(2-bromo-3,5-difluorophenyl)-1,3-dioxolane.

[0533] 1 H NMR (300 MHz, CDCl3) δ 7.20 - 7.15 (m, 1H), 6.49 - 6.87 (m, 1H), 6.08 (s, 1H), 4.17 - 4.04 (m, 4H).

[0534] Step 2: 2-(3,5-Difluoro-2-vinylphenyl)-1,3-dioxolane

[0535]

[0536] The title compound was synthesized according to a procedure similar to Step 2 of Example 1, using 2-(2-bromo-3,5-difluorophenyl)-1,3-dioxolane, to give 2-(3,5-difluoro-2-vinylphenyl)-1,3-dioxolane (3.45 g).

[0537] 1 H NMR (300 MHz, CDCl3) δ 7.20 - 7.16 (m, 1H), 6.94 - 6.83 (m, 1H), 6.82 (dd, J = 17.7, 11.7 Hz, 1H), 5.60 (dd, J = 1.2, 11.7 Hz, 1H), 5.66 - 5.60 (m, 1H), 5.95 (s, 1H), 4.18 - 4.02 (m, 4H).

[0538] Step 3: 2-(2-(2,2-Difluorocyclopropyl)-3,5-difluorophenyl)-1,3-dioxolane

[0539]

[0540] To a solution of 2-(3,5-difluoro-2-vinylphenyl)-1,3-dioxolane (from Step 2, 2.4 g, 11.31 mmol) and trimethyl(trifluoromethyl)silane (16.1 g, 113.1 mmol) in tetrahydrofuran (15 mL) was added sodium iodide (850 mg, 5.65 mmol). The resulting solution was stirred at 65 °C for 24 h. The reaction mixture was cooled to room temperature, and trimethyl(trifluoromethyl)silane (16.1 g, 113.1 mmol) and sodium iodide (850 mg, 5.65 mmol) were added. The resulting solution was stirred at 65 °C for 24 h. The reaction mixture was then partitioned between ethyl acetate and water. The organic phase was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure to afford the title compound 2-(2-(2,2-difluorocyclopropyl)-3,5-difluorophenyl)-1,3-dioxolane (1.5 g) as a brown liquid.

[0541] 1 H NMR (400 MHz, CDCl3) δ 7.26 - 7.14 (m, 1H), 6.90 - 6.77 (m, 1H), 6.04 (s, 1H), 4.17 - 4.03 (m, 4H), 2.77 - 2.66 (m, 1H), 1.98 - 1.70 (m, 2H).

[0542] Step 4: 2-(2,2-Difluorocyclopropyl)-3,5-difluorobenzaldehyde

[0543]

[0544] To a solution of 2-(2-(2,2-difluorocyclopropyl)-3,5-difluorophenyl)-1,3-dioxolane (from Step 3, 1.5 g, 5.72 mmol) in diethyl ether (15 mL) was added 6N HCl (3 mL). The resulting solution was stirred at room temperature for 3 h. This was washed with water (50 mL), saturated NaHCO3 solution (100 mL) and brine, and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica (0→5% ethyl acetate in hexanes) to afford the title compound 2-(2,2-difluorocyclopropyl)-3,5-difluorobenzaldehyde (1.25 g) as a colorless liquid.

[0545] 1 H NMR (300 MHz, CDCl3) δ 10.22 (s, 1H), 7.50 - 7.39 (m, 1H), 7.20 - 7.05 (m, 1H), 2.80 - 2.60 (m, 1H), 2.18 - 1.89 (m, 1H), 1.65 - 1.53 (m, 1H).

[0546] Step 5: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(2,2-difluorocyclopropyl)-3,5-difluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0547]

[0548] The title compound was synthesized using General Procedure 1 of Method I (using the aldehyde from Step 4 (1.0 g, 4.58 mmol), Intermediate A (862 mg, 4.58 mmol) and Intermediate B (608 mg, 4.58 mmol)) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(2,2-difluorocyclopropyl)-3,5-difluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (2.5 g, crude).

[0549] LCMS Rt = 1.683 min; MS m / z 504 [M+H]+; [Method 7]

[0550] Step 6: Methyl 4-(2-(2,2-difluorocyclopropyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0551]

[0552] The title compound was synthesized using Step 2 of General Procedure I (using the intermediate from Step 5, 2.5 g, 4.97 mmol). The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to give the title compound methyl 4-(2-(2,2-difluorocyclopropyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (120 mg) as an off-white solid. The racemate was separated into its enantiomers by chiral SFC (mobile phase: 20% IPA / CO2 80 g / min; column: Whelk-O1 SS21x250 mm; instrument: Thar80_SN4740).

[0553] Example 3

[0554] 20.9 mg of the first eluting stereoisomer (68.8%) as a white solid.

[0555] SFC Rt = 2.40 min ((SS)Whelk-O1 4.6x100 mm, 5 μm, 5%→

[0556] 55% IPA) in CO2

[0557] LCMS Rt = 2.27 min; MS m / z 416.2 [M+H]+; [Method 4]

[0558] 1 H NMR (400 MHz, methanol-d4) δ 6.93 - 6.73 (m, 2H), 5.90 - 5.77 (m, 1H), 5.77 - 5.64 (m, 1H), 5.46 (s, 1H), 4.83 (s, 2H), 3.54 (s, 3H), 3.20 - 3.02 (m, 1H), 2.09 (tdd, J = 11.8, 7.5, 5.9 Hz, 1H), 1.95 (ddt, J = 16.5, 7.8, 3.4 Hz, 1H).

[0559] Example 4

[0560] 20.6 mg of the second eluting stereoisomer as a white solid (67.8%).

[0561] SFC Rt = 2.43 min ((SS) Whelk-O1 4.6 x 100 mm, 5 μm, 5% in CO2 →

[0562] 55% IPA).

[0563] LCMS Rt = 2.21 min; MS m / z 416.4 [M+H]+; [Method 4]

[0564] 1 H NMR (400 MHz, methanol-d4) δ 6.93 - 6.72 (m, 2H), 5.74 (d, J = 3.6 Hz, 1H), 5.62 (d, J = 4.1 Hz, 1H), 5.42 (s, 1H), 5.00 - 4.90 (m, 2H), 3.60 (s, 3H), 2.97 - 2.80 (m, 1H), 2.68 - 2.49 (m, 1H), 2.02 (tdd, J = 11.9, 7.8, 5.9 Hz, 1H).

[0565] The remaining stereoisomers were separated as Examples 3b and 4b

[0566] Example 3b

[0567] 21.6 mg of the third eluting stereoisomer as a white solid (74.1%).

[0568] SFC Rt = 2.68 min ((SS) Whelk-O1 4.6 x 100 mm, 5 μm, 5% in CO2 →

[0569] 55% IPA

[0570] LCMS Rt = 2.22 min; MS m / z 416.6 [M+H]+; [Method 4]

[0571] 1 H NMR (400 MHz, methanol-d4) δ 6.78 - 6.63 (m, 2H), 5.76 - 5.64 (m, 1H), 5.64 - 5.52 (m, 1H), 5.33 (s, 1H), 4.70 (s, 2H), 3.41 (s, 3H), 3.07 - 2.91 (m, 1H), 1.96 (tdd, J = 11.8, 7.6, 5.9 Hz, 1H), 1.90 - 1.77 (m, 1H).

[0572] Example 4b

[0573] 20.6 mg of the fourth eluted stereoisomer as a white solid (70.7%).

[0574] SFC Rt = 2.79 min ((SS) Whelk-O1 4.6 x 100 mm, 5 μm, 5% in CO2 →

[0575] 55% IPA

[0576] LCMS Rt = 2.21 min; MS m / z 416.3 [M+H]+; [Method 4]

[0577] 1 H NMR (400 MHz, methanol-d4) δ 6.91 - 6.73 (m, 2H), 5.80 - 5.67 (m, 1H), 5.67 - 5.55 (m, 1H), 5.41 (d, J = 1.6 Hz, 1H), 4.98 - 4.88 (m, 2H), 3.59 (s, 3H), 2.98 - 2.81 (m, 1H), 2.68 - 2.48 (m, 1H), 2.00 (tdd, J = 11.8, 7.8, 5.9 Hz, 1H).

[0578] Example 5: Methyl (R)-4-(3,5-difluoro-2-((R or S)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0579]

[0580] Step 1: 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde

[0581]

[0582] To a solution of 2-(3,5-difluoro-2-vinylphenyl)-1,3-dioxolane (prepared in Step 2 of Example 4, 5 g, 23.58 mmol) and ruthenium chloride·XH2O (490 mg, 2.35 mmol) in dichloromethane (50 mL) and water (10 mL) was added diacetoxyiodobenzene (11.4 g, 35.37 mmol). The resulting solution was stirred at 30 °C for 2 h. The reaction mixture was filtered and washed with ethyl acetate (200 mL). The filtrate was added to water (200 mL), and the product was extracted into ethyl acetate (500 mL). The EtOAc phase was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to give the title compound 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde as a colorless liquid (3 g).

[0583] 1 1H NMR (300 MHz, CDCl3) δ 10.41 (s, 1H), 7.35 (d, J = 9.6 Hz, 1H), 6.95 - 6.85 (m, 1H), 6.53 (s, 1H), 4.08 (s, 4H).

[0584] Step 2: 1-(2-(1,3-Dioxolan-2-yl)-4,6-difluorophenyl)ethan-1-ol

[0585]

[0586] At 0 °C, to a solution of 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde (from Step 1, 1.0 g, 4.67 mmol) in dry tetrahydrofuran (10 mL) was added methylmagnesium bromide (2.33 mL, 3 M in ether, 4.67 mmol). The resulting solution was stirred at room temperature for 1 h. The reaction mixture was quenched with saturated ammonium chloride (10 mL) at 0 °C and diluted with ethyl acetate (100 mL). The organic phase was separated, washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound 1-(2-(1,3-dioxolan-2-yl)-4,6-difluorophenyl)ethan-1-ol as a colorless liquid (950 mg). The crude compound was used in the next step without further purification.

[0587] 11H NMR (300 MHz, CDCl3) δ 7.17 (dd, J = 2.4, 6.3 Hz, 1H), 6.89 - 6.72 (m, 1H), 6.17 (s, 1H), 5.31 (dd, J = 6.9, 13.5 Hz, 1H), 4.15 - 4.02 (m, 4H), 2.64 - 2.59 (m, 1H), 1.58 (d, J = 9.0 Hz, 3H).

[0588] Step 3: 2-(3,5-Difluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane

[0589]

[0590] At -78 °C, diethylaminosulfur trifluoride (1.0 g, 6.13 mmol) was added to a solution of 1-(2-(1,3-dioxolan-2-yl)-4,6-difluorophenyl)ethan-1-ol (from Step 2, 950 mg, 4.13 mmol) in dichloromethane (10 mL). The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was quenched with saturated ammonium chloride (10 mL) at 0 °C and diluted with ethyl acetate (100 mL). The organic phase was separated, washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure to afford the title compound 2-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane (600 mg) as a thick yellow liquid. The crude compound was used in the next step without further purification.

[0591] 1 1H NMR (300 MHz, CDCl3) δ 7.20 (dd, J = 2.4, 6.3 Hz, 1H), 6.87 - 6.75 (m, 1H), 6.15 - 5.98 (m, 1H), 6.11 (s, 1H), 4.15 - 4.02 (m, 4H), 1.70 (dd, J = 6.6, 22.8 Hz, 3H).

[0592] Step 4: 3,5-Difluoro-2-(1-fluoroethyl)benzaldehyde

[0593]

[0594] At room temperature, 4N HCl (2 mL) was added to a solution of 2-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane (from step 3, 600 mg, 2.58 mmol) in diethyl ether (10 mL). The resulting solution was stirred at room temperature for 3 hours. This was washed with water (50 mL), saturated NaHCO3 solution (100 mL), brine, and dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound 3,5-difluoro-2-(1-fluoroethyl)benzaldehyde (200 mg) as a colorless liquid. (Note: The aldehyde obtained is volatile in nature)

[0595] 1 1H NMR (300 MHz, CDCl3) δ 10.44 (d, J = 3.0 Hz, 1H), 7.20 (d, J = 9.3 Hz, 1H), 7.10 - 6.98 (m, 1H), 6.43 - 6.19 (m, 1H), 1.78 (dd, J = 7.2, 23.1 Hz, 3H).

[0596] Step 5: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0597]

[0598] The title compound was synthesized using step 1 of General Procedure I (using the aldehyde from step 4 (200 mg, 1.06 mmol)) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate as a mixture of diastereomers. The crude product was used in the next step without purification.

[0599] LCMS Rt = 2.272 min; MS m / z 472.3 [M-H]-; [Method 7]

[0600] Step 6: Methyl 4-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0601]

[0602] Step 2 using General Procedure I to synthesize the title compound (using the mixture of intermediates from Step 5, 800 mg, 1.75 mmol) gave methyl 4-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (2 g crude) as an off-white solid.

[0603] The diastereoisomer mixture was separated into its two isomers using combi- flash chromatography (0→80%) to give 100 mg of the first diastereoisomer as a white solid and 80 mg of the second diastereoisomer as a white solid.

[0604] Peak-1: 100 mg and Peak-2: 80 mg were further separated into their enantiomers using preparative chiral HPLC [Method 7] to obtain four isomers.

[0605] Example 5

[0606] The first eluted enantiomer obtained, 21 mg as a white solid.

[0607] Chiral HPLC Rt = 4.418 min; [Chiral Analytical Method 2]

[0608] LCMS Rt = 1.514 min; MS m / z 384.0 [M-H]-; [Method 7]

[0609] 1 1H NMR: (400 MHz, CD3OD) δ 6.87 - 6.74 (m, 2H), 6.46 - 6.18 (m, 1H), 5.75 (t, J = 1.0 Hz, 1H), 5.63 (dd, J = 1.6, 0.7 Hz, 1H), 5.20 - 5.12 (m, 1H), 4.91 - 4.81 (m, 2H), 3.53 (s, 3H), 1.83 (ddd, J = 22.7, 6.6, 1.4 Hz, 3H).

[0610] Example 5b

[0611] The second eluted enantiomer obtained, 22 mg as a white solid.

[0612] Chiral HPLC Rt = 4.792 min; [Chiral Analytical Method 2]

[0613] LCMS Rt = 1.513 min; MS m / z 384.0 [M-H]-; [Method 7]

[0614] 11H NMR (400 MHz, CD3OD) δ 6.87 - 6.74 (m, 2H), 6.46 - 6.18 (m, 1H), 5.75 (t, J = 1.0 Hz, 1H), 5.63 (dd, J = 1.6, 0.7 Hz, 1H), 5.20 - 5.12 (m, 1H), 4.91 - 4.81 (m, 2H), 3.53 (s, 3H), 1.83 (ddd, J = 22.7, 6.6, 1.4 Hz, 3H).

[0615] Example 5c

[0616] The third eluted enantiomer, 18 mg, obtained as a white solid.

[0617] Chiral HPLC Rt = 6.777 min; [Chiral analysis method 2]

[0618] LCMS Rt = 1.476 min; MS m / z 384.1 [M-H]-; [Method 7]

[0619] 1 1H NMR: (400 MHz, CDCl3) δ 6.77 - 6.63 (m, 2H), 6.48 (dd, J = 44.3, 6.6 Hz, 1H), 5.85 - 5.77 (m, 1H), 5.70 (d, J = 4.5 Hz, 1H), 5.10 (s, 1H), 4.83 - 4.77 (m, 2H), 3.56 (s, 3H), 1.79 (ddd, J = 22.5, 6.7, 1.1 Hz, 3H).

[0620] Example 5d

[0621] The fourth eluted enantiomer, 16 mg, obtained as a white solid using chiral HPLC method 7.

[0622] Chiral HPLC Rt = 7.475 min; [Chiral analysis method 2]

[0623] LCMS Rt = 2.00 min; MS m / z 384.2 [M-H]-; [Method 9]

[0624] 1 1H NMR (400 MHz, CDCl3) δ 6.77 - 6.63 (m, 2H), 6.48 (dd, J = 44.3, 6.6 Hz, 1H), 5.85 - 5.77 (m, 1H), 5.70 (d, J = 4.5 Hz, 1H), 5.10 (s, 1H), 4.83 - 4.77 (m, 2H), 3.56 (s, 3H), 1.79 (ddd, J = 22.5, 6.7, 1.1 Hz, 3H).

[0625] Example 6: Methyl (R)-4-(2-((S)-1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0626]

[0627] Step 1: 1-(2-(1,3-Dioxolan-2-yl)-6-fluorophenyl)-2-fluoroethan-1-one

[0628]

[0629] Under a nitrogen atmosphere, a solution of 2-(2-bromo-3-fluorophenyl)-1,3-dioxolane (from Step 1, Example 1, 26 g, 105.23 mmol) in THF (250 mL) was cooled to -78 °C. Then n-butyllithium in hexane solution (44.19 mL, 2.5 M, 110.49 mmol) was added, and the reaction was stirred at -78 °C for 30 min. Then ethyl 2-fluoroacetate (22.33 g, 210.47 mmol) was added to the reaction, and the resulting mixture was stirred at -78 °C for an additional 60 min. The reaction mixture was quenched with saturated ammonium chloride solution (100 mL) and extracted with ethyl acetate (2 x 250 mL). The combined organic phases were washed with water (250 mL), brine (250 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to give the title compound 1-(2-(1,3-dioxolan-2-yl)-6-fluorophenyl)-2-fluoroethan-1-one (16.2 g) as a colorless liquid.

[0630] 1 1H NMR (400 MHz, CDCl3) δ 7.45 (m, 1H), 7.37 (d, J = 7.8, 1H), 7.13 (dd, J = 9.3, 8.2 Hz, 1H), 6.02 (s, 1H), 5.23 (d, J = 1.4 Hz, 1H), 5.11 (d, J = 1.3 Hz, 1H), 4.08 - 3.87 (m, 4H).

[0631] Step 2: 1-(2-(1,3-Dioxolan-2-yl)-6-fluorophenyl)-2-fluoroethan-1-ol

[0632]

[0633] Under a nitrogen atmosphere at 0 °C, sodium borohydride (3.9 g, 103.07 mmol) was added to a stirred solution of 1-(2-(1,3-dioxolan-2-yl)-6-fluorophenyl)-2-fluoroethan-1-one (from Step 1, 19.6 g, 85.89 mmol) in methanol (100 mL). The resulting mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with ice water and extracted with ethyl acetate (2 x 250 mL). The combined organic phases were washed with water (250 mL), then with brine (250 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→30% ethyl acetate in petroleum ether) to give the title compound 1-(2-(1,3-dioxolan-2-yl)-6-fluorophenyl)-2-fluoroethan-1-ol as a colorless liquid (18 g).

[0634] 1 1H NMR (400 MHz, CDCl3) δ 7.44 (dd, J = 7.9, 1.2 Hz, 1H), 7.33 (dd, J = 8.0, 8.0 Hz, 1H), 7.10 (dd, J = 8.2, 1.3 Hz, 1H), 6.13 (s, 1H), 5.47 (m, 1H), 4.98 - 4.51 (m, 2H), 4.22 - 3.97 (m, 4H), 3.04 (m, 1H).

[0635] Step 3: 2-(2-(1,2-Difluoroethyl)-3-fluorophenyl)-1,3-dioxolane

[0636]

[0637] A solution of 1-(2-(1,3-dioxolan-2-yl)-6-fluorophenyl)-2-fluoroethan-1-ol (from Step 2, 18 g, 78.18 mmol) in dichloromethane (150 mL) was cooled to -78 °C, then diethylaminosulfur trifluoride (DAST) (15.49 mL, 117.28 mmol) was added and the resulting reaction mixture was stirred at -78 °C for 1 h. The reaction mixture was allowed to warm slowly to 0 °C and stirred for an additional 1 h. Water (150 mL) was added and the product was extracted with dichloromethane (2 x 200 mL). The combined organic phases were washed with saturated sodium bicarbonate solution (2 x 200 mL), water (200 mL), brine (200 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to give the title compound 2-(2-(1,2-difluoroethyl)-3-fluorophenyl)-1,3-dioxolane as a colorless liquid (12.4 g).

[0638] 1 1H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 7.8 Hz, 1H), 7.42 - 7.35 (m, 1H), 7.12 (dd, J = 10.6, 8.2 Hz, 1H), 6.21 (m, 1H), 6.04 (s, 1H), 5.22 - 4.85 (m, 1H), 4.75 - 4.42 (m, 1H), 4.20 - 3.97 (m, 4H).

[0639] Step 4: 2-(1,2-Difluoroethyl)-3-fluorobenzaldehyde

[0640]

[0641] At 0 °C, 2N HCl (100 mL) was added to a solution of 2-(2-(1,2-difluoroethyl)-3-fluorophenyl)-1,3-dioxolane (from Step 3, 12.4 g, 53.40 mmol) in diethyl ether (150 mL). The resulting reaction mixture was stirred at room temperature for 8 h. The reaction mixture was extracted with diethyl ether (200 mL), washed with water (2 x 100 mL), brine (100 mL), and dried over Na2SO4. The solvent was removed under reduced pressure and purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to give the title compound 2-(1,2-difluoroethyl)-3-fluorobenzaldehyde (10 g) as a colorless liquid.

[0642] 1 1H NMR (400 MHz, CDCl3) δ 10.30 (s, 1H), 7.88 - 7.70 (m, 1H), 7.56 (m, 1H), 7.44 - 7.30 (m, 1H), 6.70 - 6.36 (m, 1H), 4.95 - 4.70 (m, 2H)

[0643] Step 5: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(1,2-difluoroethyl)-3-fluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0644]

[0645] Step 1 of General Procedure I was used to synthesize the title compound (using the aldehyde from Step 4, 9 g, 47.83 mmol), to obtain the title compound 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(1,2-difluoroethyl)-3-fluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate as a mixture of diastereomers as an off-white solid (25.0 g, crude).

[0646] The crude product was used in the next step without further purification and analysis.

[0647] Step 6: Methyl 4-(2-(1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0648]

[0649] Step 2 of General Procedure I was used to synthesize the title compound (using the mixture of intermediates from Step 5, 25 g, 52.8 mmol), to obtain the title compound methyl 4-(2-(1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate as an off-white solid (1 g crude). The mixture of diastereomers was separated into its two isomers using silica gel flash chromatography (0→80%), giving 1.51 g of the first diastereomer as a white solid and 820 mg of the second diastereomer as a white solid.

[0650] The previously separated peaks of diastereomers - peak-1: 200 mg and peak-2: 200 mg were further separated into their enantiomers using preparative chiral HPLC [Method 7] to obtain four isomers.

[0651] Example 6

[0652] 56 mg of the first eluted enantiomer as a white solid was obtained.

[0653] Chiral HPLC Rt = 7.623 min; [Chiral Analysis Method 2]

[0654] LCMS Rt = 1.445 min; MS m / z 384.0 [M-H]-; [Method 7]

[0655] 11H NMR (400 MHz, CD3OD) δ 7.41 - 7.35 (m, 1H), 7.17 (dt, J = 7.9, 1.3 Hz, 1H), 6.98 (dd, J = 11.4, 8.1 Hz, 1H), 6.66 - 6.33 (m, 1H), 5.75 (d, J = 3.7 Hz, 1H), 5.68 - 5.56 (m, 1H), 5.18 (s, 1H), 5.15 - 4.91 (m, 2H), 4.85 (s, 2H), 3.52 (s, 3H).

[0656] The stereochemistry of Example 6 (methyl 2 - methyl - 5 - oxo - 4 - phenyl - 1,4,5,7 - tetrahydrofuro[3,4 - b]pyridine - 3 - carboxylate) was determined by single - crystal X - ray crystallography, and it was confirmed that the active enantiomer is the R - enantiomer ( Figure 7 ).

[0657] Example 6b

[0658] The second eluted enantiomer obtained, 54 mg as a white solid.

[0659] Chiral HPLC Rt = 8.757 min; [Chiral analytical method 2]

[0660] LCMS Rt = 1.443 min; MS m / z 386.3 [M + H]+; [Method 7]

[0661] 1 1H NMR: (400 MHz, CD3OD) δ 7.41 - 7.35 (m, 1H), 7.17 (dt, J = 7.9, 1.3, 1.3 Hz, 1H), 7.10 - 6.88 (m, 1H), 6.67 - 6.26 (m, 1H), 5.82 - 5.70 (m, 1H), 5.69 - 5.57 (m, 1H), 5.18 (s, 1H), 5.15 - 4.91 (m, 2H), 4.85 (d, J = 0.7 Hz, 2H), 3.52 (s, 3H).

[0662] Example 6c

[0663] The third eluted enantiomer obtained, 67 mg as a white solid.

[0664] Chiral HPLC = Rt = 10.574 min; [Chiral analytical method 2]

[0665] LCMS Rt = 1.379 min; MS m / z 384.0 [M - H] - ; [Method 7]

[0666] 11H NMR (400 MHz, CD3OD) δ 7.41 - 7.35 (m, 1H), 7.13 (dt, J = 7.9, 1.3, 1.3 Hz, 1H), 6.97 (dd, J = 11.3, 8.2 Hz, 1H), 6.60 (ddd, J = 47.2, 18.0, 8.4 Hz, 1H), 5.75 (s, 1H), 5.63 (s, 1H), 5.14 (s, 1H), 5.12 - 4.92 (m, 1H), 4.84 (s, 2H), 4.70 (ddd, J = 44.6, 32.7, 10.8 Hz, 1H), 3.53 (s, 3H).

[0667] Example 6d

[0668] 69 mg of the fourth eluted enantiomer as a white solid was obtained.

[0669] Chiral HPLC Rt = 13.93 min [Chiral analytical method 2]

[0670] LCMS Rt = 1.379 min; MS m / z 384.0 [M-H]-; [Method 7]

[0671] 1 1H NMR (400 MHz, CD3OD) δ 7.41 - 7.35 (m, 1H), 7.13 (dt, J = 7.8, 1.3 Hz, 1H), 6.97 (dd, J = 11.3, 8.2 Hz, 1H), 6.60 (ddd, J = 47.1, 17.9, 8.3 Hz, 1H), 5.75 (s, 1H), 5.63 (s, 1H), 5.17 - 5.11 (m, 1H), 5.11 - 4.92 (m, 1H), 4.84 (s, 2H), 4.70 (ddd, J = 45.1, 33.0, 11.1 Hz, 1H), 3.53 (s, 3H).

[0672] Example 7: Methyl (R)-4-(2-((R or S)-1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[0673]

[0674] Step 1: 3-Ethoxycyclopent-2-en-1-one

[0675]

[0676] At room temperature, pTSA (193 mg, 1.019 mmol) and EtOH (22.61 ml, 387.35 mmol) were added to a stirred solution of cyclopentane-1,3-dione (5.0 g, 50.96 mmol) in toluene (70 mL). The resulting mixture was stirred at 120 °C for 10 h using a Dean-Stark apparatus. The solvent was removed under reduced pressure to give the crude compound. The crude product was purified by flash chromatography on silica (0→50% ethyl acetate in petroleum ether) to afford the title compound 3-ethoxycyclopent-2-en-1-one as a brown solid (4.3 g).

[0677] 1 H NMR (400 MHz, CDCl3) δ 5.26 (s, 1H), 4.02 (q, J = 6.6 Hz, 2H), 2.60 - 2.55 (m, 2H), 2.44 - 2.40 (m, 2H), 1.39 (t, J = 7.2 Hz, 3H).

[0678] Step 2: 3-Aminocyclopent-2-en-1-one

[0679]

[0680] At room temperature, ammonium hydroxide solution (25 mL, 387.35 mmol) was added to a stirred solution of 3-ethoxycyclopent-2-en-1-one (from Step 1, 4.3 g, 34.08 mmol) in ethanol (50 mL). The resulting mixture was stirred at 85 °C for 16 h. The solvent was removed under reduced pressure to give the title compound 3-aminocyclopent-2-en-1-one as a brown solid (3.2 g).

[0681] LCMS Rt = 0.114 min; MS m / z 98.2 [M+H]+; [Method 1]

[0682] Step 3: Methyl 4-(2-(1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[0683]

[0684] To a solution of aldehyde (from Example 6, Step 4, 3.0 g, 15.944 mmol) was added methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 2.54 g, 19.133 mmol) and cyclopentane-1,3-dione (1.56 g, 15.944 mmol) in tert-butanol (25 mL). The reaction mixture was stirred at 80 °C for 48 h. The solvent was removed under reduced pressure to give the crude compound. The crude product was purified by flash chromatography on silica gel (0→70% ethyl acetate in petroleum ether) to give the title compound methyl 4-(2-(1,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate (500 mg, crude) as a mixture of diastereomers. The mixture of diastereomers was separated into its two isomers using flash chromatography on silica gel (0→80%), giving 12 mg of the first diastereomer as a white solid and 15 mg of the second diastereomer as a white solid.

[0685] The peaks of the first separated diastereomer, peak-1: 12 mg and peak-2: 15 mg, were further separated into their enantiomers using preparative chiral HPLC [Method 7] to obtain four isomers.

[0686] Example 7

[0687] The first eluted enantiomer, 2 mg, obtained as a white solid.

[0688] Chiral HPLC Rt = 9.084 min; [Chiral Analytical Method 4]

[0689] LCMS Rt = 1.477 min; MS m / z 384.05 [M+H]+; [Method 2]

[0690] 1 1H NMR (400 MHz, methanol-d4) δ 7.36 - 7.24 (m, 1H), 7.06 (d, J = 7.8 Hz, 1H), 6.92 (dd, J = 11.4, 8.2 Hz, 1H), 6.87 - 6.60 (m, 1H), 5.72 (s, 1H), 5.60 (s, 1H), 5.05 (s, 1H), 4.69 (ddd, J = 44.8, 32.9, 10.8 Hz, 2H), 3.54 (s, 3H), 2.70 (t, J = 5.0 Hz, 2H), 2.43 - 2.29 (m, 2H).

[0691] Example 7b

[0692] The second eluted enantiomer, 4 mg, obtained as a white solid.

[0693] Chiral HPLC Rt = 10.439 min; [Chiral analysis method 4]

[0694] LCMS Rt = 1.477 min; MS m / z 384.2 [M+H]+; [Method 2]

[0695] 1 H NMR (400 MHz, methanol-d4) δ 7.37 - 7.23 (m, 1H), 7.10 - 7.02 (m, 1H), 6.92 (dd, J = 11.4, 8.1 Hz, 1H), 6.86 - 6.60 (m, 1H), 5.72 (s, 1H), 5.60 (s, 1H), 5.05 (s, 1H), 4.69 (ddd, J = 44.9, 33.5, 11.1 Hz, 2H), 3.54 (s, 3H), 2.76 - 2.66 (m, 2H), 2.42 - 2.27 (m, 2H).

[0696] Example 7c

[0697] 4 mg of the third eluted enantiomer obtained as a white solid.

[0698] Chiral HPLC Rt = 12.453 min; [Chiral analysis method 4]

[0699] LCMS Rt = 1.327 min; MS m / z 384.2 [M+H]+; [Method 2]

[0700] 1 H NMR (400 MHz, methanol-d4) δ 7.32 (tdd, J = 8.1, 5.6, 1.5 Hz, 1H), 7.08 (dt, J = 7.8, 1.3 Hz, 1H), 6.93 (dd, J = 11.5, 8.1 Hz, 1H), 6.60 - 6.38 (m, 1H), 5.81 - 5.69 (m, 1H), 5.69 - 5.54 (m, 1H), 5.13 - 5.07 (m, 2H), 5.07 - 4.93 (m, 1H), 3.53 (s, 3H), 2.75 - 2.67 (m, 2H), 2.45 - 2.22 (m, 2H).

[0701] Example 7d

[0702] 4 mg of the fourth eluted enantiomer obtained as a white solid.

[0703] Chiral HPLC Rt = 13.173 min; [Chiral analysis method 4]

[0704] LCMS Rt = 1.33 min; MS m / z 384.2 [M+H]+; [Method 2]

[0705] 1 1H NMR (400 MHz, methanol-d4) δ 7.40 - 7.25 (m, 1H), 7.14 - 7.05 (m, 1H), 6.93 (dd, J = 11.4, 8.2 Hz, 1H), 6.60 - 6.38 (m, 1H), 5.80 - 5.66 (m, 1H), 5.66 - 5.53 (m, 1H), 5.13 - 5.07 (m, 2H), 5.07 - 4.94 (m, 1H), 3.53 (s, 3H), 2.71 (t, J = 4.8 Hz, 2H), 2.44 - 2.21 (m, 2H).

[0706] Example 8: Methyl (R)-4-(3-fluoro-2-((R or S)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0707]

[0708] Step 1: 1-(2-(1,3-Dioxolan-2-yl)-6-fluorophenyl)ethan-1-ol

[0709]

[0710] Under a nitrogen atmosphere, a solution of 2-(2-bromo-3-fluorophenyl)-1,3-dioxolane (from Step 1, Example 1, 30 g, 121.42 mmol) in THF (300 mL) was cooled to -78 °C. Then n-butyllithium (58.3 mL, 2.5 M, 147.71 mmol) in a hexane solution was added over 10 min, and the resulting mixture was stirred at -78 °C for 1 h. Acetaldehyde (6.42 g, 145.71 mmol) was added, and the resulting mixture was stirred at -78 °C for 1 h. The reaction mixture was quenched with saturated ammonium chloride solution (50 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with water (50 mL), brine (50 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to give the title compound 1-(2-(1,3-dioxolan-2-yl)-6-fluorophenyl)ethan-1-ol (15 g) as a colorless liquid.

[0711] 11H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.0 Hz, 1H), 7.30 - 7.18 (m, 1H), 7.10 - 7.01 (m, 1H), 6.15 (s, 1H), 5.30 - 5.25 (m, 1H), 4.15 - 3.95 (m, 4H), 2.75 - 2.69 (m, 1H), 1.58 (dd, J = 6.6, 22.8 Hz, 3H).

[0712] Step 2: 2-(3-Fluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane

[0713]

[0714] At -78 °C, diethylaminosulfur trifluoride (19.3 mL, 141.37 mmol) was added to a solution of 1-(2-(1,3-dioxolan-2-yl)-6-fluorophenyl)ethan-1-ol (from Step 1, 15 g, 70.68 mmol) in dichloromethane (150 mL). The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was quenched with saturated ammonium chloride (20 mL) at 0 °C and diluted with ethyl acetate (100 mL). The organic phase was separated, washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to give the title compound 2-(3-fluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane (8.5 g) as a colorless liquid.

[0715] 1 1H NMR (300 MHz, CDCl3) δ 7.38 (d, J = 7.5 Hz, 1H), 7.30 - 7.18 (m, 1H), 7.13 - 7.01 (m, 1H), 6.20 - 5.95 (m, 1H), 6.10 (s, 1H), 4.16 - 4.02 (m, 4H), 1.74 (dd, J = 7.2, 23.1 Hz, 3H).

[0716] Step 3: 3-Fluoro-2-(1-fluoroethyl)benzaldehyde

[0717]

[0718] At room temperature, 4N HCl (85 mL) was added to a solution of 2-(3-difluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane (from Step 2, 8.5 g, 69.38 mmol) in diethyl ether (150 mL). The resulting solution was stirred at room temperature for 3 hours. This was washed with water (100 mL), saturated NaHCO3 solution (200 mL), brine, and dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound 3-difluoro-2-(1-fluoroethyl)benzaldehyde (6.5 g) as a colorless liquid. (Note: The aldehyde obtained is volatile in nature).

[0719] 1 1H NMR (300 MHz, CDCl3) δ 10.44 (d, J = 1.0 Hz, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.45 - 7.42 (m, 1H), 7.35 - 7.30 (m, 1H), 6.45 - 6.38 (m, 1H), 1.92 - 1.72 (m, 3H).

[0720] Step 4: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0721]

[0722] The title compound was synthesized using Step 1 of General Procedure I (using the aldehyde from Step 3, 10 g, 58.76 mmol) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (13 g, crude) as a mixture of diastereomers.

[0723] LCMS Rt = 1.795 min; MS m / z 454.2 [M-H]-; [Method 9]

[0724] Step 5: Methyl 4-(3-fluoro-2-(1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0725]

[0726] The title compound, methyl 4-(3-fluoro-2-(1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (5 g, crude), was obtained as an off-white solid by the synthesis of the title compound using Step 2 of General Procedure I (using a mixture of intermediates from Step 4 (13.0 g, 28.54 mmol)). The diastereoisomer mixture was separated by flash chromatography on silica (0→80% ethyl acetate in hexanes) to give 1.04 g of the first diastereoisomer and 1.45 g of the second diastereoisomer.

[0727] The diastereoisomers were further separated using chiral SFC: column 2.1 x 25.0 cm Chiralcel OX-H; CO2 co-solvent (solvent B), methanol isocratic method 25% co-solvent, 80 g / min, system pressure 100 bar

[0728] Example 8

[0729] 37 mg of the title compound as the first eluting stereoisomer

[0730] LCMS Rt = 1.95 min; MS m / z 366.3 [M-H]-; [Method 4]

[0731] SFC Rt = 1.12 min; mobile phase: 20% MeOH (0.1% isopropylamine) in CO2, 4 mL / min; column: Chiralcel OX-H 4.6 x 100 mm

[0732] 1 1H NMR (400 MHz, DMSO-d6) δ 10.12 (d, J = 3.3 Hz, 1H), 7.34 (qd, J = 7.0, 2.9 Hz, 1H), 7.21 - 6.91 (m, 2H), 6.35 (dq, J = 45.6, 6.6 Hz, 1H), 5.87 - 5.45 (m, 2H), 5.03 (s, 1H), 4.86 (d, J = 2.8 Hz, 2H), 3.44 (s, 3H), 1.80 (dd, J = 22.9, 6.6 Hz, 3H).

[0733] Example 8b

[0734] 38 mg as the second eluting stereoisomer

[0735] LCMS Rt = 1.95 min; MS m / z 366.3 [M-H]-; [Method 4]

[0736] SFC Rt = 1.52 min; Mobile phase: 20% MeOH (0.1% isopropylamine) in CO2, 4 mL / min; Column: Chiralcel OX-H 4.6 x 100 mm

[0737] 1 1H NMR: (400 MHz, DMSO-d6) δ 10.12 (d, J = 3.3 Hz, 1H), 7.56 - 7.23 (m, 1H), 7.25 - 6.94 (m, 2H), 6.35 (dd, J = 45.5, 6.9 Hz, 1H), 5.66 (dd, J = 47.9, 2.7 Hz, 2H), 5.03 (s, 1H), 4.86 (d, J = 2.6 Hz, 2H), 3.44 (s, 3H), 1.80 (dd, J = 22.8, 6.5 Hz, 3H).

[0738] Example 8c

[0739] 42 mg as the third eluting stereoisomer

[0740] LCMS Rt = 1.88 min; MS m / z 366.3 [M-H]-; [Method 4]

[0741] SFC Rt = 2.08 min; Mobile phase: 20% MeOH (0.1% isopropylamine) in CO2, 4 mL / min; Column: Chiralcel OX-H 4.6 x 100 mm

[0742] 1 1H NMR (400 MHz, DMSO-d6) δ 10.13 (d, J = 3.3 Hz, 1H), 7.33 (td, J = 8.0, 5.4 Hz, 1H), 7.23 - 6.92 (m, 2H), 6.49 (dd, J = 44.7, 6.8 Hz, 1H), 5.68 (d, J = 47.8 Hz, 2H), 4.97 (s, 1H), 4.83 (s, 2H), 3.44 (s, 3H), 1.70 (dd, J = 22.7, 6.6 Hz, 3H).

[0743] Example 8d

[0744] 40 mg: as the fourth eluting stereoisomer

[0745] LCMS Rt = 1.88 min; MS m / z 366.3 [M-H]-; [Method 4]

[0746] SFC Rt = 3.01 min; Mobile phase: 20% MeOH (0.1% isopropylamine) in CO2, 4 mL / min; Column: Chiralcel OX-H 4.6 x 100 mm

[0747] 1 1H NMR: (400 MHz, DMSO-d6) δ 10.13 (d, J = 3.4 Hz, 1H), 7.33 (td, J = 8.0, 5.4 Hz, 1H), 7.20 - 6.93 (m, 2H), 6.69 - 6.31 (m, 1H), 5.68 (d, J = 47.8 Hz, 2H), 4.97 (s, 1H), 4.83 (s, 2H), 3.44 (s, 3H), 1.70 (dd, J = 22.8, 6.5 Hz, 3H).

[0748] The stereochemistry of Example 8 (methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate) was determined by single crystal x-ray crystallography and the active enantiomer was confirmed to be the R-enantiomer ( Figure 8 ).

[0749] Example 9: Methyl (R)-4-(2-(difluoromethoxy)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0750]

[0751] Step 1: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(difluoromethoxy)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0752]

[0753] The title compound was synthesized using Step 1 of General Procedure I (using 2-(difluoromethoxy)benzaldehyde (350 mg, 2.033 mmol), Intermediate A (382.3 mg, 2.033 mmol) and Intermediate B (275.65 mg, 2.033 mmol)) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(difluoromethoxy)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (600 mg, crude). Without analysis, it was used in the next step.

[0754] Step 2: Methyl 4-(2-(difluoromethoxy)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0755]

[0756] To the crude intermediate from Step 1 (600 mg, 1.30 mmol) in methanol (20 mL) was added potassium hydroxide (293.44 mg, 5.22 mmol). The resulting solution was stirred at 65 °C for 30 min. Water was added to the reaction mixture, and the solid was precipitated and filtered. The solid was washed with diethyl ether and collected and dried under reduced pressure to give the title compound methyl 4-(2-(difluoromethoxy)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (100 mg).

[0757] Using chiral HPLC [Method 4], 90 mg of the racemic mixture was separated into its enantiomers.

[0758] Example 9

[0759] 40 mg of the first eluted enantiomer as a white solid, 44% yield.

[0760] Chiral HPLC Rt = 6.481 min; [Chiral analytical method 1]

[0761] LCMS Rt = 1.473 min; MS m / z 370 [M+H]+; [Method 7]

[0762] 1 1H NMR (400 MHz, CDCl3) δ 7.30 (dd, J = 7.5, 1.9 Hz, 1H), 7.24 - 7.12 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H), 6.81 - 6.43 (m, 1H), 5.82 - 5.69 (m, 1H), 5.68 - 5.56 (m, 1H), 5.19 (s, 1H), 4.77 (d, J = 1.6 Hz, 2H), 3.55 (d, J = 0.7 Hz, 3H). No exchangeable NH proton was seen.

[0763] The stereochemistry of Example 9 (methyl (R)-4-(2-(difluoromethoxy)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate) was determined by single crystal x-ray crystallography, and it was confirmed that the active enantiomer is the R-enantiomer ( Figure 9 ).

[0764] Example 9b

[0765] The second eluted enantiomer as a white solid, 38 mg, 42% yield.

[0766] Chiral HPLC Rt = 10.303 min; [Chiral analysis method 1]

[0767] LCMS Rt = 1.475 min; MS m / z 370.1 [M+H]+; [Method 7]

[0768] 1 1H NMR: (400 MHz, CDCl3) δ 7.30 (dd, J = 7.5, 1.9 Hz, 1H), 7.24 - 7.12 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H), 6.81 - 6.43 (m, 1H), 5.82 - 5.69 (m, 1H), 5.68 - 5.56 (m, 1H), 5.19 (s, 1H), 4.77 (d, J = 1.6 Hz, 2H), 3.55 (d, J = 0.7 Hz, 3H).

[0769] Example 10: Methyl (R)-4-(3,5-difluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0770]

[0771] Step 1: 1-Bromo-3,5-difluoro-2-(trifluoromethyl)benzene

[0772]

[0773] A solution of 3,5-difluoro-2-(trifluoromethyl)aniline (10 g, 50.735 mmol) in 48% HBr / H2O aqueous solution (80 mL / 80 mL) was cooled to -5 °C, then NaNO2 (8.74 g in 100 mL H2O, 126.83 mmol) was slowly added over a 5 min period, and the resulting mixture was stirred at -5 °C for 1 h. At -5 °C, the reaction mixture was treated portionwise with CuBr (10.92 g, 76.103 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 4 h. The reaction mixture was extracted with hexane (3 X 200 mL). The organic phases were combined, washed with saturated brine solution (100 mL), and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica (in petroleum ether) to give the title compound 1-bromo-3,5-difluoro-2-(trifluoromethyl)benzene (8 g) as a colorless liquid.

[0774] 1 1H NMR (400 MHz, CDCl3) δ 7.31 (dt, J = 7.6, 2.2 Hz, 1H), 6.95 - 6.88 (m, 1H).

[0775] Step 2: 1,5 - Difluoro - 2 - (trifluoromethyl) - 3 - vinylbenzene

[0776]

[0777] To a solution of 1 - bromo - 3,5 - difluoro - 2 - (trifluoromethyl)benzene (from Step 1, 12.0 g, 45.99 mmol) and trifluoro(vinyl)-14 - borane in isopropanol (120 mL) were added potassium salt (12.3 g, 91.98 mmol) and triethylamine (19.16 mL, 137.98 mmol). The resulting solution was purged with argon for 10 min, after which [1,1′ - bis(diphenylphosphino)ferrocene]dichloropalladium(II).DCM (3.75 g, 4.599 mmol) was added. The reaction mixture was stirred at 80 °C for 6 h. The reaction mixture was filtered through a Celite pad and washed with ethyl acetate (100 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (in petroleum ether) to afford the title compound 1,5 - difluoro - 2 - (trifluoromethyl) - 3 - vinylbenzene as a colorless liquid (6.5 g).

[0778] 1 1H NMR (300 MHz, CDCl3) δ 7.16 - 6.97 (m, 2H), 6.85 - 6.80 (m, 1H), 5.67 (d, J = 17.2 Hz, 1H), 5.49 (d, J = 10.9 Hz, 1H).

[0779] Step 3: 3,5 - Difluoro - 2 - (trifluoromethyl)benzaldehyde

[0780]

[0781] 1,5 - Difluoro - 2 - (trifluoromethyl) - 3 - vinylbenzene (from Step 2, 6.5 g, 31.23 mmol) and RuCl 3.XH2O (647 mg, 3.123 mmol) was added to a solution of diacetoxyiodobenzene (20 g, 62.46 mmol) in dichloromethane:H2O (130 mL:30 mL). The resulting solution was stirred at room temperature for 2 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with dichloromethane (200 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→5% ethyl acetate in petroleum ether) to afford the title compound 3,5-difluoro-2-(trifluoromethyl)benzaldehyde (3.0 g) as a colorless liquid. 1 1H NMR (300 MHz, CDCl3) δ 10.34 (m, 1H), 7.82 - 7.52 (m, 1H), 7.22 - 7.07 (m, 1H).

[0782] Step 4: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(3,5-difluoro-2-(trifluoromethyl)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0783]

[0784] The title compound was synthesized using Step 1 of General Procedure I (using the aldehyde from Step 3 (400 mg, 1.903 mmol)) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(3,5-difluoro-2-(trifluoromethyl)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (900 mg, crude).

[0785] LCMS Rt = 1.76 min; MS m / z 496.0 [M+H]+; [Method 7]

[0786] Step 5: Methyl 4-(3,5-difluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0787]

[0788] Step 2 using general procedure I to synthesize the title compound (using the intermediate from step 4 (900 mg, 1.8 mmol)). The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to give the title compound methyl 4-(3,5-difluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (90 mg) as an off-white solid. The racemic mixture was separated into its enantiomers using chiral HPLC [Method 7].

[0789] Example 10

[0790] The first eluted enantiomer, 30 mg, obtained as a white solid.

[0791] Chiral HPLC Rt = 5.502 min; [Chiral analytical method 2]

[0792] LCMS Rt = 1.542 min; MS m / z 406 [M-H]-; [Method 7]

[0793] 1 H NMR: (400 MHz, CDCl3) δ 7.30 (d, J = 7.2 Hz, 1H), 6.87 (d, J = 9.5 Hz, 1H), 6.85 6.75 (m, 1H), 5.79 (t, J = 1.2, 1.2 Hz, 1H), 5.67 (t, J = 1.1 Hz, 1H), 5.62 (s, 1H), 4.80 (d, J = 1.2 Hz, 2H), 3.54 (s, 3H).

[0794] Example 10b

[0795] The second eluted enantiomer, 30 mg, obtained as a white solid.

[0796] Chiral HPLC Rt = 6.935 min; [Chiral analytical method 1]

[0797] LCMS Rt = 1.542 min; MS m / z 406 [M-H]-; [Method 7]

[0798] 1 H NMR: (400 MHz, CDCl3) δ 7.30 (d, J = 7.2 Hz, 1H), 6.87 (d, J = 9.5 Hz, 1H), 6.85 6.75 (m, 1H), 5.79 (t, J = 1.2, 1.2 Hz, 1H), 5.67 (t, J = 1.1 Hz, 1H), 5.62 (s, 1H), 4.80 (d, J = 1.2 Hz, 2H), 3.54 (s, 3H).

[0799] Example 11: Methyl (R)-4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0800]

[0801] Step 1: 2-(1,3-Dioxolan-2-yl)-6-fluorobenzaldehyde

[0802]

[0803] Under a nitrogen atmosphere, a solution of 2-(2-bromo-3-fluorophenyl)-1,3-dioxolane (from Step 1, Example 1, 7 g, 28.45 mmol) in THF (70 mL) was cooled to -78 °C. Then n-butyllithium (13.66 mL, 2.5 M, 58.5 mmol) in a hexane solution was added over 10 min, and the resulting mixture was stirred at -78 °C for 45 min. DMF (2.5 g, 34.15 mmol) was added, and the resulting mixture was stirred at -78 °C for 1.15 h. The reaction mixture was quenched with saturated ammonium chloride solution (50 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with water (50 mL), then with brine (50 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to give the title compound 2-(1,3-dioxolan-2-yl)-6-fluorobenzaldehyde (5 g) as a colorless liquid.

[0804] 1 1H NMR (300 MHz, CDCl3) δ 10.52 (s, 1H), 7.67 - 7.53 (m, 2H), 7.22 - 7.12 (m, 1H), 6.50 (s, 1H), 4.27 - 3.99 (m, 4H).

[0805] Step 2: 2-(2-(2,2-Difluorovinyl)-3-fluorophenyl)-1,3-dioxolane

[0806]

[0807] A solution of acetonitrile (65 mL), triphenylphosphine (28.1 g, 107.1 mmol), potassium iodide (11.85 g, 71.4 mmol) and 2-(1,3-dioxolan-2-yl)-6-fluorobenzaldehyde (from Step 1, 7 g, 35.7 mmol) was heated to 70 °C under a nitrogen atmosphere and stirred for 30 min. Methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (12 g, 62.4 mmol) was added slowly over a 10 min period (the color of the mass turned yellow during the addition). The resulting mixture was stirred at 70 °C for an additional 3 h, then cooled to room temperature and diluted with diethyl ether. The precipitated solid was filtered and washed with diethyl ether (100 mL). The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→5% ethyl acetate in petroleum ether) to afford the title compound 2-(2-(2,2-difluorovinyl)-3-fluorophenyl)-1,3-dioxolane (6.9 g) as a pale yellow liquid.

[0808] 1 1H NMR: (300 MHz, CDCl3) δ 7.39 (dd, J = 7.8, 1.4 Hz, 1H), 7.31 (m, 1H), 7.11 (m, 1H), 5.87 (s, 1H), 5.39 (m, 1H), 4.21 - 3.95 (m, 4H).

[0809] Step 3: 2-(2-(2,2-Difluoroethyl)-3-fluorophenyl)-1,3-dioxolane

[0810]

[0811] To a solution of 2-(2-(2,2-difluorovinyl)-3-fluorophenyl)-1,3-dioxolane (from Step 2, 3 g, 13.04 mmol) in ethyl acetate (60 mL) was added 10% Pd-C (1 g). The resulting reaction mixture was kept in a Parr-Shaker at 60 psi under a hydrogen atmosphere at room temperature for 48 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with ethyl acetate (30 mL). The filtrate was concentrated under reduced pressure and the crude product was purified by flash chromatography on silica (0→30% ethyl acetate in petroleum ether) to afford the title compound 2-(2-(2,2-difluoroethyl)-3-fluorophenyl)-1,3-dioxolane (3 g) as a colorless liquid.

[0812] 11H NMR (300 MHz, CDCl3) δ 7.42 - 7.35 (m, 1H), 7.34 - 7.27 (m, 1H), 7.15 - 7.08 (m, 1H), 6.31 - 5.78 (m, 2H), 4.19 - 4.02 (m, 4H), 3.38 (m, 2H).

[0813] Step 4: 2-(2,2-difluoroethyl)-3-fluorobenzaldehyde

[0814]

[0815] To a solution of 2-(2-(2,2-difluoroethyl)-3-fluorophenyl)-1,3-dioxolane (from Step 3, 3 g, 12.93 mmol) in diethyl ether (40 mL) was added 6N HCl (5 mL). The resulting solution was stirred at room temperature for 3 h. This was washed with water (50 mL), saturated NaHCO3 solution (100 mL), brine, and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (0→5% ethyl acetate in petroleum ether) to give the title compound 2-(2,2-difluoroethyl)-3-fluorobenzaldehyde (1.9 g) as a colorless liquid.

[0816] 1 1H NMR (400 MHz, CDCl3) δ 10.14 (d, J = 1.6 Hz, 1H), 7.70 - 7.63 (m, 1H), 7.52 (td, J = 8.0, 7.9, 5.2 Hz, 1H), 7.35 - 7.30 (m 1H), 6.03 (m, 1H), 3.83 - 3.58 (m, 2H).

[0817] Step 5: 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0818]

[0819] The title compound was synthesized using Step 1 of General Procedure I (using the aldehyde from Step 4, 400 mg, 2.12 mmol) to give the title compound 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (1 g, crude).

[0820] LCMS Rt = 1.764 min; MS m / z 473.4 [M+H]+; [Method 7]

[0821] Step 6: Methyl 4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0822]

[0823] The title compound was synthesized using Step 2 of General Procedure I (using the intermediate from Step 5, 1 g, 2.11 mmol). The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to give the title compound, methyl 4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, as an off-white solid (85 mg). The racemic mixture was separated into its enantiomers using chiral preparative HPLC [Method 6].

[0824] Example 11

[0825] The first eluted enantiomer, obtained as a white solid (30 mg).

[0826] Chiral HPLC Rt = 6.325 min; [Chiral Analytical Method 2]

[0827] LCMS Rt = 1.508 min; MS m / z 386.0 [M+H]+; [Method 7]

[0828] 1 1H NMR (400 MHz, CDCl3) δ 7.34 - 7.27 (m, 1H), 7.21 (m, 1H), 7.01 - 6.89 (m, 2H), 6.55 - 6.17 (m, 1H), 5.81 (dd, J = 1.7, 0.8 Hz, 1H), 5.69 (t, J = 1.1 Hz, 1H), 5.08 (s, 1H), 4.78 (d, J = 0.9 Hz, 2H), 3.94 - 3.73 (m, 1H), 3.60 - 3.55 (m, 1H), 3.54 (s, 3H).

[0829] Example 11b

[0830] The first eluted enantiomer, obtained as a white solid (30 mg).

[0831] Chiral HPLC Rt = 8.402 min; [Chiral Analytical Method 2]

[0832] LCMS Rt = 1.508 min; MS m / z 386.4 [M+H]+; [Method 7]

[0833] 1 1H NMR (400 MHz, CDCl3) δ 7.34 - 7.27 (m, 1H), 7.21 (td, J = 8.0, 8.0, 5.6 Hz, 1H), 7.01 - 6.89 (m, 2H), 6.55 - 6.17 (m, 1H), 5.81 (dd, J = 1.7, 0.8 Hz, 1H), 5.69 (t, J = 1.1, 1.1 Hz, 1H), 5.08 (s, 1H), 4.78 (d, J = 0.9 Hz, 2H), 3.94 - 3.73 (m, 1H), 3.60 - 3.55 (m, 1H), 3.54 (s, 3H).

[0834] Example 12: Methyl (R)-4-(2-(2,2-difluoroethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0835]

[0836] Step 2: 2-(3,5-Difluoro-2-vinylphenyl)-1,3-dioxolane

[0837]

[0838] A solution of 2-(2-bromo-3,5-difluorophenyl)-1,3-dioxolane (from Step 1, Example 3, 5 g, 18.94 mmol), potassium vinyltrifluoroborate (5.8 g, 37.89 mmol), and triethylamine (7.91 mL, 56.82 mmol) in isopropanol (50 mL) was degassed with argon for 10 min, and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).DCM (1.55 g, 1.89 mmol) was added. The resulting solution was degassed with argon again for 10 min and stirred at 80 °C for 5 h. The reaction mixture was filtered and washed with ethyl acetate (100 mL). The filtrate was dissolved in water (1 L) and extracted into ethyl acetate (2 L). The EtOAc was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to give the title compound 2-(3,5-difluoro-2-vinylphenyl)-1,3-dioxolane (3.45 g) as a colorless liquid. Note: The compound is volatile in nature and hexane is present in the compound.

[0839] 11H NMR (300 MHz, CDCl3) δ 7.20 - 7.16 (m, 1H), 6.94 - 6.83 (m, 1H), 6.82 (dd, J = 17.7, 11.7 Hz, 1H), 5.60 (dd, J = 1.2, 11.7 Hz, 1H), 5.66 - 5.60 (m, 1H), 5.95 (s, 1H), 4.18 - 4.02 (m, 4H).

[0840] Step 3: 2-(1,3-Dioxolan-2-yl)-4,6-difluorobenzaldehyde

[0841]

[0842] To a solution of 2-(3,5-difluoro-2-ethenylphenyl)-1,3-dioxolane (from Step 2, 5 g, 23.58 mmol) and ruthenium chloride·XH2O (490 mg, 2.35 mmol) in dichloromethane (50 mL) and water (10 mL) was added (diacetoxyiodo)benzene (11.4 g, 35.37 mmol). The resulting solution was stirred at 30 °C for 2 h. The reaction mixture was filtered and washed with ethyl acetate (200 mL). The filtrate was dissolved in water (200 mL) and the product was extracted into ethyl acetate (500 mL). The organic layer was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to give the title compound 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde as a colorless liquid (3 g). Note: The compound is volatile in nature and hexane is present in the compound.

[0843] 1 1H NMR (300 MHz, CDCl3) δ 10.41 (s, 1H), 7.35 (d, J = 9.6 Hz, 1H), 6.95 - 6.85 (m, 1H), 6.53 (s, 1H), 4.08 (s, 4H).

[0844] Step 4: 2-(2-(2,2-Difluorovinyl)-3,5-difluorophenyl)-1,3-dioxolane

[0845]

[0846] Under a nitrogen atmosphere, triphenylphosphine (1.83 g, 7.0 mmol), potassium iodide (775 mg, 4.66 mmol), and 2-(1,3-dioxolan-2-yl)-6-fluorobenzaldehyde (from Step 3, 500 mg, 2.33 mmol) were added to acetonitrile (4.5 mL). The reaction was stirred at 70 °C for 30 min. Then, 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde (783 mg, 4.07 mmol) was slowly added over a 10-min period (the color of the material turned yellow during the addition), and the resulting mixture was stirred at 70 °C for an additional 3 h. The reaction mixture was then cooled to room temperature and diluted with diethyl ether, the precipitated solid was filtered, and washed with diethyl ether (100 mL). The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→5% ethyl acetate in petroleum ether) to give the title compound 2-(2-(2,2-difluorovinyl)-3,5-difluorophenyl)-1,3-dioxolane (400 mg) as a pale yellow liquid.

[0847] 1 1H NMR (300 MHz, CDCl3) δ 7.19 - 7.12 (m, 1H), 6.89 - 6.80 (m, 1H), 5.85 (d, J = 0.9 Hz, 1H), 5.31 (dt, J = 26.1, 1.5 Hz, 1H), 4.16 - 3.98 (m, 4H).

[0848] Step 5: 2-(2-(2,2-Difluoroethyl)-3,5-difluorophenyl)-1,3-dioxolane

[0849]

[0850] To a solution of 2-(2-(2,2-difluorovinyl)-3,5-difluorophenyl)-1,3-dioxolane (from Step 4, 400 mg, 1.61 mmol) in ethyl acetate (10 mL) was added 10% Pd-C (180 mg). The resulting reaction mixture was kept in a Parr shaker at room temperature under a hydrogen atmosphere at a pressure of 60 psi for 48 h. The reaction mixture was filtered through a pad of diatomaceous earth and washed with ethyl acetate (30 mL). The filtrate was concentrated under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→30% ethyl acetate in petroleum ether) to give the title compound 2-(2-(2,2-difluoroethyl)-3,5-difluorophenyl)-1,3-dioxolane (400 mg) as a colorless liquid.

[0851] 11H NMR (300 MHz, CDCl3) δ 7.22 - 7.12 (m, 1H), 6.81 - 6.75 (m, 1H), 6.26 - 5.75 (m, 2H), 4.20 - 3.99 (m, 4H), 3.36 - 3.33 (m, 2H).

[0852] Step 6: 2-(2,2-Difluoroethyl)-3,5-difluorobenzaldehyde

[0853]

[0854] To a solution of 2-(2-(2,2-difluoroethyl)-3,5-difluorophenyl)-1,3-dioxolane (from Step 5, 400 mg, 1.6 mmol) in diethyl ether (10 mL) was added 5N HCl (1 mL). The resulting solution was stirred at room temperature for 3 h. This was washed with water (50 mL), saturated NaHCO3 solution (100 mL) and brine, and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→5% ethyl acetate in petroleum ether) to give the title compound 2-(2,2-difluoroethyl)-3,5-difluorobenzaldehyde (250 mg) as a colorless liquid.

[0855] 1 1H NMR (300 MHz, CDCl3) δ 10.11 (d, J = 1.3 Hz, 1H), 7.51 - 7.34 (m, 1H), 7.20 - 7.07 (m, 1H), 6.31 - 5.73 (m, 1H), 3.79 - 3.56 (m, 2H).

[0856] Step 7: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(2,2-difluoroethyl)-3,5-difluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0857]

[0858] The title compound was synthesized using Step 1 of General Procedure I (using the aldehyde from Step 6, 250 mg, 1.21 mmol) to give the title compound 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-(2,2-difluoroethyl)-3,5-difluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (800 mg, crude).

[0859] LCMS Rt = 1.77 min; MS m / z 491.0 [M+]+; [Method 7]

[0860] Step 8: Methyl 4-(2-(2,2-difluoroethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0861]

[0862] The title compound was synthesized using Step 2 of General Procedure I (using the intermediate from Step 7, 800 mg, 1.62 mmol). The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to afford the title compound, methyl 4-(2-(2,2-difluoroethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (44 mg) as an off-white solid. The racemic mixture was separated into its enantiomers using chiral preparative HPLC [Method 6].

[0863] Example 12

[0864] The first eluted enantiomer, 11 mg, was obtained as a white solid.

[0865] Chiral HPLC Rt = 5.674 min; [Chiral Analytical Method 2]

[0866] LCMS Rt = 1.561 min; MS m / z 403.9 [M+]+; [Method 7]

[0867] 1 1H NMR (400 MHz, CD3OD) δ 6.87 - 6.76 (m, 2H), 6.64 - 6.26 (m, 1H), 5.76 (s, 1H), 5.64 (s, 1H), 5.13 (d, J = 1.4 Hz, 1H), 4.87 - 4.83 (m, 2H), 3.50 - 3.36 (m, 1H), 3.70 (s, 2H), 3.53 (s, 3H).

[0868] Example 12b

[0869] The second eluted enantiomer, 10 mg, was obtained as a white solid.

[0870] Chiral HPLC Rt = 7.215 min; [Chiral Analytical Method 2]

[0871] LCMS Rt = 1.557 min; MS m / z 403.9 [M+]+; [Method 7]

[0872] 11H NMR (400 MHz, CD3OD) δ 6.87 - 6.76 (m, 2H), 6.64 - 6.26 (m, 1H), 5.76 (s, 1H), 5.64 (s, 1H), 5.13 (d, J = 1.4 Hz, 1H), 4.87 - 4.83 (m, 2H), 3.50 - 3.36 (m, 1H), 3.70 (s, 2H), 3.53 (s, 3H).

[0873] Example 13: Methyl (R)-4-(2-cyclopropyl-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0874]

[0875] Step 1: 2-Cyclopropyl-3,5-difluorobenzaldehyde

[0876]

[0877] To a solution of 2-bromo-3,5-difluorobenzaldehyde (10 g, 45.20 mmol) and cyclopropylboronic acid (4.67 g, 54.2 mmol) in toluene (100 mL) was added 2N K2CO3 (102 mL, 204 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).DCM (3.17 g, 3.88 mmol). The resulting solution was degassed with argon for 10 min and stirred at 100 °C for 4 h. The reaction mixture was filtered and washed with ethyl acetate (200 mL). The filtrate was added to water (500 mL), and the product was extracted into ethyl acetate (2 L). The EtOAc phase was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica (0→1% ethyl acetate in petroleum ether) to give the title compound 2-cyclopropyl-3,5-difluorobenzaldehyde (8 g) as a colorless liquid.

[0878] 1 1H NMR (300 MHz, CDCl3) δ 10.71 (d, J = 3 Hz, 1H), 7.40 - 730 (m, 1H), 7.05 - 6.90 (m, 1H), 2.05 - 1.95 (m, 1H), 1.19 - 1.09 (m, 2H), 0.85 - 0.75 (m, 2H).

[0879] Step 2: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(2-cyclopropyl-3,5-difluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0880]

[0881] The title compound was synthesized by General Procedure 1 of General Procedure I (using the aldehyde from Step 1 (1.8 g, 9.88 mmol)) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(2-cyclopropyl-3,5-difluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (4.61 g, crude).

[0882] LCMS Rt = 1.586 min; MS m / z 477.0 [M+H]+; [Method 10]

[0883] Step 3: Methyl 4-(2-cyclopropyl-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0884]

[0885] The title compound was synthesized by Step 2 of General Procedure I (using the intermediate from Step 2, 4.61 g, 9.871 mmol). The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to give the title compound, methyl 4-(2-cyclopropyl-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, as an off-white solid (350 mg).

[0886] The racemic mixture was separated into its enantiomers using chiral preparative HPLC [Method 9].

[0887] Example 13

[0888] The first eluted enantiomer, 135 mg, was obtained as a white solid.

[0889] Chiral HPLC Rt = 5.33 min; [Chiral Analytical Method 2]

[0890] LCMS Rt = 1.591 min; MS m / z 380.2 [M+H]+; [Method 7]

[0891] 11H NMR (300 MHz, CD3OD) δ 6.78 - 6.70 (m, 1H), 6.66 (m, 1H), 5.75 (s, 1H), 5.61 (d, J = 14.1 Hz, 2H), 4.84 (q, J = 1.7, 1.7, 0.9 Hz, 2H), 3.53 (s, 3H), 2.08 (m, 1H), 1.18 (m, 1H), 1.07 - 0.87 (m, 2H), 0.75 (m, 1H).

[0892] Example 14: Methyl (R)-4-(2-cyclopropyl-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[0893]

[0894] Step 2: Methyl 4-(2-cyclopropyl-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[0895]

[0896] To a solution of the aldehyde (from Step 1, Example 13, 300 mg, 1.64 mmol) was added methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 220 mg, 1.64 mmol) and cyclopentane-1,3-dione (161 mg, 1.64 mmol) in ethanol (5 mL). The reaction mixture was stirred at 80 °C for 16 h. The solvent was removed under reduced pressure to give the crude compound. The crude product was purified by flash chromatography on silica (0→70% ethyl acetate in petroleum ether) to afford the title compound methyl 4-(2-cyclopropyl-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate (55 mg) as an off-white solid. The racemic mixture was separated into its enantiomers using chiral preparative HPLC [Method 3].

[0897] Example 14

[0898] The first eluting enantiomer, 17 mg, obtained as a white solid.

[0899] Chiral HPLC Rt = 8.588 min; [Chiral Analytical Method 3]

[0900] LCMS Rt = 1.548 min; MS m / z 378.0 [M+H]+; [Method 7]

[0901] 11H NMR (400 MHz, CDCl3) δ 7.21 (d, J = 7.2 Hz, 1H), 6.65 - 6.48 (m, 2H), 5.78 (d, J = 0.9 Hz, 1H), 5.65 (d, J = 0.8 Hz, 1H), 5.60 - 5.52 (m, 1H), 3.56 (s, 3H), 2.75 - 2.56 (m, 2H), 2.52 - 2.32 (m, 2H), 2.20 - 2.13 (m, 1H), 1.34 - 1.13 (m, 1H), 1.05 - 0.98 (m, 2H), 0.83 - 0.69 (m, 1H).

[0902] Example 14b

[0903] 17 mg of the second eluted enantiomer obtained as a white solid.

[0904] Chiral HPLC Rt = 11.651 min; [Chiral analysis method 3]

[0905] LCMS Rt = 1.548 min; MS m / z 378.0 [M+H]+; [Method 7]

[0906] 1 1H NMR (400 MHz, CDCl3) δ 7.21 (d, J = 7.2 Hz, 1H), 6.65 - 6.48 (m, 2H), 5.78 (d, J = 0.9 Hz, 1H), 5.65 (d, J = 0.8 Hz, 1H), 5.60 - 5.52 (m, 1H), 3.56 (s, 3H), 2.75 - 2.56 (m, 2H), 2.52 - 2.32 (m, 2H), 2.20 - 2.13 (m, 1H), 1.34 - 1.13 (m, 1H), 1.05 - 0.98 (m, 2H), 0.83 - 0.69 (m, 1H).

[0907] Example 15: Methyl (R)-4-(2-(difluoromethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0908]

[0909] Step 1: 2-(Difluoromethyl)-1-fluoro-3-vinylbenzene

[0910]

[0911] To a solution of 1-bromo-2-(difluoromethyl)-3-fluorobenzene (2.0 g, 8.889 mmol) was added potassium vinyltrifluoroborate (2.38 g, 17.78 mmol) and triethylamine (3.7 mL, 26.66 mmol) in isopropanol (20 mL). The resulting solution was purged with argon for 10 min and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).DCM (725 mg, 0.889 mmol) was added. The reaction mixture was stirred at 80 °C for 6 h. The reaction mixture was filtered through a Celite pad and washed with ethyl acetate (100 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (in petroleum ether) to give the title compound 2-(difluoromethyl)-1-fluoro-3-vinylbenzene (1.2 g) as a colorless liquid.

[0912] 1 H NMR (400 MHz, CDCl3) δ 7.48 - 7.40 (m, 1H), 7.30 - 7.15 (m, 1H), 7.04 (t, J = 53.4 Hz, 1H), 7.04 - 6.90 (m, 2H), 5.44 (d, J = 11.2 Hz, 1H), 5.70 (d, J = 11.2 Hz, 1H).

[0913] Step 2: 2-(Difluoromethyl)-3-fluorobenzaldehyde

[0914]

[0915] To a solution of 2-(difluoromethyl)-1-fluoro-3-vinylbenzene (from Step 1, 1.2 g, 6.970 mmol) was added RuCl 3. XH2O (145 mg, 0.697 mmol) and diacetoxyiodobenzene (6.73 g, 20.91 mmol) in dichloromethane:H2O (20 mL:6 mL). The resulting solution was stirred at room temperature for 2 h. The reaction mixture was filtered through a Celite pad and washed with dichloromethane (200 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→5% ethyl acetate in petroleum ether) to give the title compound 2-(difluoromethyl)-3-fluorobenzaldehyde (500 mg) as a colorless liquid.

[0916] 11H NMR (400 MHz, CDCl3) δ 10.42 (t, J = 2.0 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 7.70 - 7.59 (m, 1H), 7.45 - 7.35 (m, 1H), 7.30 (t, J = 53.2 Hz, 1H).

[0917] Step 3: 3 - Ethyl 5 - methyl 2 - (acetoxymethyl) - 4 - (2 - (difluoromethyl) - 3 - fluorophenyl) - 6 - (fluoromethyl) - 1,4 - dihydropyridine - 3,5 - dicarboxylate

[0918]

[0919] The title compound was synthesized using Step 1 of General Procedure I (using the aldehyde from Step 2, 500 mg, 2.871 mmol) to give 3 - ethyl 5 - methyl 2 - (acetoxymethyl) - 4 - (2 - (difluoromethyl) - 3 - fluorophenyl) - 6 - (fluoromethyl) - 1,4 - dihydropyridine - 3,5 - dicarboxylate (1.5 g, crude).

[0920] LCMS Rt = 1.716 min; MS m / z 460.1 [M + H]+; [Method 7]

[0921] Step 4: Methyl 4 - (2 - (difluoromethyl) - 3 - fluorophenyl) - 2 - (fluoromethyl) - 5 - oxo - 1,4,5,7 - tetrahydrofuro[3,4 - b]pyridine - 3 - carboxylate

[0922]

[0923] The title compound was synthesized using Step 2 of General Procedure I (using the intermediate from Step 3, 1.0 g, 2.177 mmol). The crude product was purified by flash chromatography on silica gel (0 → 50% ethyl acetate in petroleum ether) to give the title compound, methyl 4 - (2 - (difluoromethyl) - 3 - fluorophenyl) - 2 - (fluoromethyl) - 5 - oxo - 1,4,5,7 - tetrahydrofuro[3,4 - b]pyridine - 3 - carboxylate, as an off - white solid (280 mg). The racemic mixture was separated into its enantiomers using chiral preparative HPLC [Method 5].

[0924] Example 15

[0925] The first eluted enantiomer, 90 mg, as a white solid, was obtained.

[0926] Chiral HPLC Rt = 10.05 min; [Chiral Analytical Method 2]

[0927] LCMS Rt = 1.947 min; MS m / z 372.2 [M+H]+; [Method 9]

[0928] 1 1H NMR (400 MHz, CDCl3) δ 7.44 - 7.28 (m, 2H), 7.09 - 6.96 (m, 2H), 5.80 (d, J = 0.8 Hz, 1H), 5.68 (d, J = 0.9 Hz, 1H), 5.20 (s, 1H), 4.80 (d, J = 1.1 Hz, 2H), 3.54 (s, 3H). No exchangeable NH protons were observed in the spectrum.

[0929] Example 15b

[0930] 90 mg of the second eluted enantiomer as a white solid was obtained.

[0931] Chiral HPLC Rt = 12.05 min; [Chiral Analytical Method 2]

[0932] LCMS Rt = 2.09 min; MS m / z 372.2 [M+H]+; [Method 9]

[0933] 1 1H NMR (400 MHz, CDCl3) δ 7.43 - 7.33 (m, 2H), 7.10 - 6.93 (m, 2H), 5.80 (d, J = 0.8 Hz, 1H), 5.68 (d, J = 0.9 Hz, 1H), 5.20 (s, 1H), 4.80 (d, J = 0.9 Hz, 2H), 3.54 (s, 3H).

[0934] Example 16: Methyl (R)-4-(2-((R or S)-1,2-difluoroethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0935]

[0936] Step 1: 2-(2-Bromo-3,5-difluorophenyl)-1,3-dioxolane

[0937]

[0938] The title compound was synthesized according to a procedure similar to Step 1 of Example 1, using 15 g of 2-bromo-3,5-difluorobenzaldehyde to obtain 15 g of the desired product 2-(2-bromo-3,5-difluorophenyl)-1,3-dioxolane.

[0939] 11H NMR (300 MHz, CDCl3) δ 7.23 - 7.18 (m, 1H), 6.95 - 6.90 (m, 1H), 6.08 (t, J = 1.1 Hz, 1H), 4.24 - 3.99 (m, 4H).

[0940] Step 2: (3 - Bromo - 4 - (1,3 - dioxolan - 2 - yl) - 2,6 - difluorophenyl)trimethylsilane

[0941]

[0942] At - 78 °C, under an inert atmosphere, to a solution of 2 - (2 - bromo - 3,5 - difluorophenyl) - 1,3 - dioxolane (from Step 1, 15.0 g, 56.59 mmol) in freshly distilled THF (150 mL) was added lithium diisopropylamide (31.1 mL, 2.0 M in hexanes, 62.25 mmol), and the mixture was stirred for 30 min. Trimethylsilyl chloride (7.37 g, 67.91 mmol) was added to the reaction mixture, and the mixture was stirred at - 78 °C for 1 h. The reaction mixture was quenched with saturated NH4Cl solution (250 mL), and the product was extracted into ethyl acetate (2 X 500 mL). The combined EtOAc phases were washed with brine solution (250 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica (in petroleum ether (100%)) to give the title compound (3 - bromo - 4 - (1,3 - dioxolan - 2 - yl) - 2,6 - difluorophenyl)trimethylsilane (9.0 g) as a colorless liquid.

[0943] 1 1H NMR (300 MHz, CDCl3) δ 7.08 (dd, J = 8.9, 1.6 Hz, 1H), 6.07 (d, J = 1.3 Hz, 1H), 4.33 - 3.89 (m, 4H), 0.37 (t, J = 1.6 Hz, 9H).

[0944] Step 3: 1 - (6 - (1,3 - dioxolan - 2 - yl) - 2,4 - difluoro - 3 - (trimethylsilyl)phenyl) - 2 - fluoroethan - 1 - one

[0945]

[0946] At -78 °C, under an inert atmosphere, n-butyllithium (6.0 mL, 2.5 M in hexanes, 14.961 mmol) was slowly added to a solution of (3-bromo-4-(1,3-dioxolan-2-yl)-2,6-difluorophenyl)trimethylsilane (from step 2, 5.0 g, 14.961 mmol) in freshly distilled THF (50 mL). Then ethyl 2,2-difluoroacetate (3.1 g, 29.9 mmol) was added to the reaction mixture and stirred at -78 °C for 1 h. The reaction was quenched with saturated NH4Cl solution (250 mL) and extracted into ethyl acetate (2 X 200 mL). The combined EtOAc phases were washed with brine solution (50 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to give the title compound 1-(6-(1,3-dioxolan-2-yl)-2,4-difluoro-3-(trimethylsilyl)phenyl)-2-fluoroethan-1-one (2.5 g) as a colorless liquid.

[0947] 1 H NMR (300 MHz, CDCl3) δ 7.04 (dt, J = 8.9, 0.8 Hz, 1H), 6.02 (s, 1H), 5.22 (d, J = 1.6 Hz, 1H), 5.07 (d, J = 1.6 Hz, 1H), 4.03 - 3.79 (m, 4H), 0.37 (s, 9H).

[0948] Step 4: 1-(6-(1,3-Dioxolan-2-yl)-2,4-difluoro-3-(trimethylsilyl)phenyl)-2-fluoroethan-1-ol

[0949]

[0950] At 0 °C, sodium borohydride (594.2 mg, 15.70 mmol) was added portionwise to a solution of 1-(6-(1,3-dioxolan-2-yl)-2,4-difluoro-3-(trimethylsilyl)phenyl)-2-fluoroethan-1-one (from step 3, 2.5 g, 7.854 mmol) in methanol (20 mL). The temperature was allowed to slowly rise to room temperature over 1 h and stirred for an additional 2 h at room temperature. The reaction mixture was quenched with saturated ammonium chloride solution (50 mL) and extracted into ethyl acetate (200 mL). The EtOAc was washed with brine solution (50 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→30% ethyl acetate in petroleum ether) to give the title compound 1-(6-(1,3-dioxolan-2-yl)-2,4-difluoro-3-(trimethylsilyl)phenyl)-2-fluoroethan-1-ol (2.5 g) as a colorless liquid.

[0951] 1 H NMR (400 MHz, CDCl3) δ 7.12 (dd, J = 9.4, 1.2 Hz, 1H), 6.15 (s, 1H), 5.42 - 5.35 (m, 1H), 4.93 - 4.47 (m, 2H), 4.19 - 3.88 (m, 4H), 2.96 (dd, J = 6.1, 2.8 Hz, 1H), 0.36 (s, 9H).

[0952] Step 5: (3-(1,2-Difluoroethyl)-4-(1,3-dioxolan-2-yl)-2,6-difluorophenyl)trimethylsilane

[0953]

[0954] At 0 °C, diethylaminosulfur trifluoride (DAST) (1.24 mL, 9.36 mmol) was added to a solution of 1-(6-(1,3-dioxolan-2-yl)-2,4-difluoro-3-(trimethylsilyl)phenyl)-2-fluoroethan-1-ol (from step 4, 2.0 g, 6.24 mmol) in dichloromethane (20 mL). The reaction was allowed to warm to room temperature and stirred for 1 h. Water (50 mL) was added to the reaction mixture and the product was extracted into dichloromethane (3 X 50 mL). The DCM phases were combined, washed with saturated NaHCO3 solution (50 mL), brine, and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→2% ethyl acetate in petroleum ether) to give the title compound (3-(1,2-difluoroethyl)-4-(1,3-dioxolan-2-yl)-2,6-difluorophenyl)trimethylsilane (1.1 g) as a colorless liquid.

[0955] 1 1H NMR (400 MHz, CDCl3) δ 7.23 - 7.04 (m, 1H), 6.24 - 6.05 (m, 1H), 6.04 (s, 1H), 5.15 - 4.89 (m, 1H), 4.70 - 4.39 (m, 1H), 4.14 - 3.99 (m, 4H), 0.36 (s, 9H).

[0956] Step 6: 2-(1,2-Difluoroethyl)-3,5-difluoro-4-(trimethylsilyl)benzaldehyde

[0957]

[0958] To a solution of (3-(1,2-difluoroethyl)-4-(1,3-dioxolan-2-yl)-2,6-difluorophenyl)trimethylsilane (from Step 5, 1.1 g, 3.41 mmol) in diethyl ether (10 mL) was added 6N HCl (11 mL). The resulting solution was stirred at room temperature for 3 h. This was washed with water (50 mL), saturated NaHCO3 solution (100 mL) and brine, and dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound 2-(1,2-difluoroethyl)-3,5-difluoro-4-(trimethylsilyl)benzaldehyde (900 mg) as a colorless liquid. The product was used in the next step without further purification and analysis.

[0959] Step 7: 2-(1,2-Difluoroethyl)-3,5-difluorobenzaldehyde

[0960]

[0961] To a solution of (2-(1,2-difluoroethyl)-3,5-difluoro-4-(trimethylsilyl)benzaldehyde (from Step 6, 900 mg, 3.23 mmol) in THF (20 mL) was added TBAF (8.0 mL, 1.0 M in THF, 8.08 mmol). The resulting solution was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc (50 mL), and this was washed with water (50 mL), saturated NaHCO3 solution (100 mL) and brine, and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (5% → 10% ethyl acetate in petroleum ether) to give the title compound 2-(1,2-difluoroethyl)-3,5-difluorobenzaldehyde (400 mg) as a colorless liquid.

[0962] 11H NMR (600 MHz, CDCl3) δ 10.41 - 10.14 (m, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.15 - 7.06 (m, 1H), 6.34 - 6.30 (m, 1H), 4.95 - 4.67 (m, 2H).

[0963] Step 8: 3 - ethyl 5 - methyl 2 - (acetoxymethyl) - 4 - (2 - (1,2 - difluoroethyl) - 3,5 - difluorophenyl) - 6 - (fluoromethyl) - 1,4 - dihydropyridine - 3,5 - dicarboxylate

[0964]

[0965] The title compound was synthesized using Step 1 of General Procedure I (using the aldehyde from Step 7, 490 mg, 2.377 mmol) to give 3 - ethyl 5 - methyl 2 - (acetoxymethyl) - 4 - (2 - (1,2 - difluoroethyl) - 3,5 - difluorophenyl) - 6 - (fluoromethyl) - 1,4 - dihydropyridine - 3,5 - dicarboxylate (1.0 g, crude). Without further purification and analysis, the product was used in the next step.

[0966] Step 9: Methyl 4 - (2 - (1,2 - difluoroethyl) - 3,5 - difluorophenyl) - 2 - (fluoromethyl) - 5 - oxo - 1,4,5,7 - tetrahydrofuro[3,4 - b]pyridine - 3 - carboxylate

[0967]

[0968] The title compound was synthesized using Step 2 of General Procedure I (using the intermediate from Step 8, 1.0 g, 2.03 mmol). The crude product was purified by flash chromatography on silica gel ((0 → 50%) ethyl acetate in petroleum ether) to give the title compound methyl 4 - (2 - (1,2 - difluoroethyl) - 3,5 - difluorophenyl) - 2 - (fluoromethyl) - 5 - oxo - 1,4,5,7 - tetrahydrofuro[3,4 - b]pyridine - 3 - carboxylate (1.0 g, crude) as an off - white solid. The mixture of diastereomers was separated into its two isomers using flash chromatography on silica gel (0 → 80%) to give 70 mg of the first diastereomer as a white solid and 50 mg of the second diastereomer as a white solid. The peaks of the first separated diastereomers - peak - 1: 70 mg and peak - 2: 50 mg were further separated into their enantiomers using preparative chiral SFC [Method 8] to obtain four isomers.

[0969] Example 16

[0970] The second eluted enantiomer obtained, 25 mg as a white solid

[0971] Chiral HPLC Rt = 12.95 min; [Chiral analysis method 3]

[0972] LCMS Rt = 1.52 min; MS m / z 402.2 [M-H]-; [Method 7]

[0973] 1 H NMR (600 MHz, CDCl3) δ 7.29 (d, J = 6.8 Hz, 1H), 6.80 - 6.69 (m, 2H), 6.41 (dd, J = 48.5, 17.2 Hz, 1H), 5.80 (s, 1H), 5.72 (s, 1H), 5.36 - 4.69 (m, 5H), 3.59 (s, 3H).

[0974] Example 16b

[0975] The first eluted enantiomer, 25 mg, as a white solid

[0976] Chiral HPLC RT 10.54 min; [Chiral analysis method 3]

[0977] LCMS Rt = 1.52 min; MS m / z 402.0 [M-H]-; [Method 7]

[0978] 1 H NMR (600 MHz, CDCl3) δ 7.29 (d, J = 6.8 Hz, 1H), 6.80 - 6.69 (m, 2H), 6.41 (dd, J = 48.5, 17.2 Hz, 1H), 5.80 (s, 1H), 5.72 (s, 1H), 5.36 - 4.69 (m, 5H), 3.59 (s, 3H).

[0979] Example 16c

[0980] The third eluted enantiomer, 15 mg, as a white solid

[0981] Chiral HPLC Rt = 14.54 min; [Chiral analysis method 3]

[0982] LCMS Rt = 1.472 min; MS m / z 402.0 [M-H]-; [Method 7]

[0983] 11H NMR (400 MHz, CD3OD) δ 6.92 - 6.80 (m, 2H), 6.70 - 6.48 (m, 1H), 5.76 (s, 1H), 5.64 (s, 1H), 5.48 (s, 1H), 5.16 (s, 1H), 5.15 - 4.51 (m, 4H), 3.55 (s, 3H).

[0984] Example 16d

[0985] 15 mg of the fourth eluted enantiomer as a white solid

[0986] Chiral HPLC Rt = 18.97 min; [Chiral analysis method 3]

[0987] LCMS Rt = 1.466 min; MS m / z 402.2 [M-H]−; [Method 7]

[0988] 1 1H NMR (400 MHz, CD3OD) δ 6.92 - 6.80 (m, 2H), 6.70 - 6.48 (m, 1H), 5.76 (s, 1H), 5.64 (s, 1H), 5.48 (s, 1H), 5.16 (s, 1H), 5.15 - 4.51 (m, 4H), 3.55 (s, 3H).

[0989] Example 17: Methyl (R)-4-(3-fluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0990]

[0991] Step 1: 3-Ethyl 5-methyl 2-(acetoxymethyl)-4-(3-fluoro-2-(trifluoromethyl)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0992]

[0993] The title compound was synthesized using General Procedure I (using commercially available 3-fluoro-2-(trifluoromethyl)benzaldehyde, 1.4 g, 7.28 mmol) to give 3-ethyl 5-methyl 2-(acetoxymethyl)-4-(3-fluoro-2-(trifluoromethyl)phenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (2 g, crude). The product was used in the next step without further purification and analysis.

[0994] Step 2: Methyl 4-(3-fluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[0995]

[0996] The title compound (from Step 2, 2 g, 4.18 mmol) was synthesized using Step 2 of General Procedure I. The crude product was purified by flash chromatography on silica gel (0→70% ethyl acetate in petroleum ether) to afford the title compound, methyl 4-(3-fluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, as an off-white solid (240 mg). The racemic mixture was separated into its enantiomers using preparative chiral HPLC [Method 5].

[0997] Example 17

[0998] 80 mg of the first eluting enantiomer as a white solid, 33% yield.

[0999] Chiral HPLC Rt = 7.549 min; [Chiral Analytical Method 1]

[1000] LCMS Rt = 1.485 min; MS m / z 390.1 [M+H]+; [Method 7]

[1001] 1 1H NMR (600 MHz, CD3OD) δ 7.62 - 7.46 (m, 1H), 7.35 - 7.30 (m, 1H), 7.20 - 7.02 (m, 1H), 5.75 - 5.69 (m, 1H), 5.68 - 5.60 (m, 1H), 5.58 - 5.50 (m, 1H), 4.84 (d, J = 5.1 Hz, 2H), 3.47 (s, 3H).

[1002] Example 18: Methyl (R)-4-(2-(difluoromethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1003]

[1004] Step 1: 2-(2-Bromo-3,5-difluorophenyl)-1,3-dioxolane

[1005]

[1006] The title compound was synthesized according to a procedure similar to Step 1 of Example 1, using 15 g of 2-bromo-3,5-difluorobenzaldehyde, to obtain 15.4 g of 2-(2-bromo-3,5-difluorophenyl)-1,3-dioxolane.

[1007] 1 1H NMR (300 MHz, CDCl3) δ 7.20 - 7.15 (m, 1H), 6.49 - 6.87 (m, 1H), 6.08 (s, 1H), 4.17 - 4.04 (m, 4H).

[1008] Step 2: 2-(3,5-difluoro-2-ethenylphenyl)-1,3-dioxolane

[1009]

[1010] The title compound was synthesized according to a procedure similar to Step 2 of Example 1, using 2-(2-bromo-3,5-difluorophenyl)-1,3-dioxolane, to give 2-(3,5-difluoro-2-ethenylphenyl)-1,3-dioxolane (3.0 g).

[1011] 1 1H NMR (300 MHz, CDCl3) δ 7.20 - 7.16 (m, 1H), 6.94 - 6.83 (m, 1H), 6.82 (dd, J = 17.7, 11.7 Hz, 1H), 5.60 (dd, J = 1.2, 11.7 Hz, 1H), 5.66 - 5.60 (m, 1H), 5.95 (s, 1H), 4.18 - 4.02 (m, 4H).

[1012] Step 3: 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde

[1013]

[1014] The title compound was synthesized according to a procedure similar to Step 1 of Example 5, to give 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde (1.0 g).

[1015] 1 1H NMR (300 MHz, CDCl3) δ 10.41 (s, 1H), 7.35 (d, J = 9.6 Hz, 1H), 6.95 - 6.85 (m, 1H), 6.53 (s, 1H), 4.08 (s, 4H).

[1016] Step 4: 2-(2-(difluoromethyl)-3,5-difluorophenyl)-1,3-dioxolane

[1017]

[1018] The title compound was synthesized according to a procedure similar to Step 3 of Example 5 to give 2-(2-(difluoromethyl)-3,5-difluorophenyl)-1,3-dioxolane (570 mg).

[1019] 1 H NMR (300 MHz, CDCl3) δ 7.29 - 7.25 (m, 1H), 7.06 (t, J = 1.1 Hz, 1H), 6.95 - 6.84 (m, 1H), 6.13 (t, J = 1.2 Hz, 1H), 4.30 - 3.86 (m, 4H).

[1020] Step 5: 2-(Difluoromethyl)-3,5-difluorobenzaldehyde

[1021]

[1022] At 0 °C, 6N HCl (10 mL) was added to a solution of 2-(2-(difluoromethyl)-3,5-difluorophenyl)-1,3-dioxolane (from Step 4, 570 mg, 2.41 mmol) in diethyl ether (10 mL). The resulting reaction mixture was stirred at room temperature for 6 h. The reaction mixture was extracted into diethyl ether (50 mL) and washed with water (30 mL). The organic phase was washed with saturated NaHCO3 solution (30 mL), brine (30 mL), and dried over Na2SO4. The solvent was removed under reduced pressure to give the crude compound 2-(difluoromethyl)-3,5-difluorobenzaldehyde (360 mg) as a colorless liquid.

[1023] 1 H NMR (300 MHz, CDCl3) δ 10.42 (s, 1H), 7.74 - 7.54 (m, 1H), 7.25 (dd, J = 46.8, 10.4 Hz, 1H), 7.15 - 7.10 (m, 1H).

[1024] Step 6: 3-Ethyl 5-methyl 2-(acetyloxymethyl)-4-(2-(difluoromethyl)-3,5-difluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate

[1025]

[1026] The title compound was synthesized using Step 1 of General Procedure I (using the aldehyde from Step 5, 360 mg, 1.87 mmol) to give 3-ethyl 5-methyl 2-(acetyloxymethyl)-4-(2-(difluoromethyl)-3,5-difluorophenyl)-6-(fluoromethyl)-1,4-dihydropyridine-3,5-dicarboxylate (720 mg, crude).

[1027] LCMS Rt = 1.698 min; MS m / z 476.1 [M-H]⁻; [Method 7]

[1028] Step 7: Methyl 4-(2-(difluoromethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1029]

[1030] The title compound was synthesized using Step 2 of General Procedure I (using the intermediate from Step 6, 720 mg, 1.50 mmol). The crude product was purified by flash chromatography on silica gel (0→70% ethyl acetate in petroleum ether) to give the title compound, methyl 4-(2-(difluoromethyl)-3,5-difluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, as an off-white solid (120 mg). The racemic mixture was separated into its enantiomers using chiral preparative HPLC [Method 7].

[1031] Example 18

[1032] The first eluted enantiomer, obtained as a white solid (30 mg), 25% yield.

[1033] Chiral HPLC Rt = 17.629 min; [Chiral Analytical Method 1]

[1034] LCMS Rt = 1.454 min; MS m / z 388.2 [M-H]⁻; [Method 7]

[1035] 1 ¹H NMR (400 MHz, CD₃OD) δ 7.40 - 7.38 (m, 1H), 6.93 (dd, J = 10.3, 7.6 Hz, 2H), 5.77 (d, J = 0.8 Hz, 1H), 5.65 (d, J = 0.7 Hz, 1H), 5.27 (d, J = 1.6 Hz, 1H), 4.89 - 4.86 (m, 2H), 3.51 (s, 3H).

[1036] Example 18b

[1037] The second eluted enantiomer, obtained as a white solid (36 mg), 30% yield.

[1038] Chiral HPLC Rt = 19.634 min; [Chiral Analytical Method 1]

[1039] LCMS Rt = 1.454 min; MS m / z 388.0 [M-H]⁻; [Method 7]

[1040] 1 H NMR (400 MHz, CD₃OD) δ 7.40 - 7.38 (m, 1H), 6.93 (dd, J = 10.3, 7.6 Hz, 2H), 5.77 (d, J = 0.8 Hz, 1H), 5.65 (d, J = 0.7 Hz, 1H), 5.27 (d, J = 1.6 Hz, 1H), 4.89 - 4.86 (m, 2H), 3.51 (s, 3H).

[1041] Example 19: Methyl (R)-4-(2-(difluoromethoxy)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1042]

[1043] The title compound was prepared using General Procedure II, using commercially available 2-(difluoromethoxy)-3-fluorobenzaldehyde (249 mg, 1.311 mmol). The crude product was purified by automated flash column chromatography, eluting on a 24 g Si-column with EtOAc in heptane (gradient 0 - 100%) and further purified by mass-directed reverse-phase preparative chromatography; conditions: Xbridge C18 OBD 30x50 mm 5um column, ACN / H₂O w / NH₃OH 5 mL / min, injection 1.5 mL. The racemate was separated into its individual enantiomers using chiral SFC (mobile phase: 5% - 25% MeOH in CO₂ at 80 g / min; column: (RR)Whelk-O1 21x250 mm).

[1044] Example 19

[1045] 8.6 mg of the second eluting enantiomer (4%)

[1046] Chiral SFC Rt = 2.61 min (mobile phase: 5% - 55% MeOH, column: (RR)Whelk-O1 4.6x100 mm 5um column, 6 min run)

[1047] LCMS Rt = 1.94 min; MS m / z 388.2 [M+H]⁺; [Method 4]

[1048] 11H NMR (400 MHz, dichloromethane-d2) δ 7.46 (d, J = 6.8 Hz, 1H), 7.29 - 7.05 (m, 3H), 6.93 (t, J = 74.16 Hz, 1H), 5.88 - 5.56 (m, 2H), 5.23 (s, 1H), 4.81 (s, 2H), 3.57 (s, 3H).

[1049] Example 19b

[1050] 9.5 mg of the first eluted enantiomer (4%)

[1051] Chiral SFC Rt = 2.41 min (mobile phase: 5% - 55% MeOH column: (RR)Whelk-O1 4.6 x 100 mm 5um column, 6 min run)

[1052] LCMS Rt = 1.90 min; MS m / z 388.2 [M+H]+; [Method 4]

[1053] 1 1H NMR (400 MHz, dichloromethane-d2) δ 7.46 (d, J = 6.8 Hz, 1H), 7.29 - 7.05 (m, 3H), 6.93 (t, J = 74.16 Hz, 1H), 5.88 - 5.56 (m, 2H), 5.23 (s, 1H), 4.81 (s, 2H), 3.57 (s, 3H).

[1054] Example 20: Methyl (R)-4-(2-(difluoromethoxy)-5-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1055]

[1056] The title compound was prepared using General Procedure II, using commercially available 2-(difluoromethoxy)-5-fluorobenzaldehyde (229 mg, 1.206 mmol). The crude product was purified by automated flash column chromatography, eluting on a 24 g Si-column with EtOAc in heptane (gradient 0 - 100%) and further purified by mass-directed reverse-phase preparative chromatography; conditions: Xbridge C18 OBD 30 x 50 mm 5um column ACN / H2O w / NH3OH 5 mL / min injection 1.5 mL. The racemate was separated into its individual enantiomers using chiral SFC (mobile phase: 5% - 25% MeOH in CO2 80 g / min; column: (RR)Whelk-O1 21 x 250 mm).

[1057] Example 20

[1058] The second eluted enantiomer of 10 mg

[1059] Chiral SFC Rt = 2.51 min (Mobile phase: 5%-55% MeOH Column: (RR) Whelk-O1 4.6x100 mm 5um column, run for 6 min)

[1060] LCMS Rt = 1.91 min; MS m / z 388.1 [M+H]+; [Method 4]

[1061] 1 H NMR (400 MHz, DMSO-d6) δ 10.11 (d, J = 3.3 Hz, 1H), 7.32 - 6.85 (m, 4H), 5.79 - 5.65 (m, 1H), 5.65 - 5.48 (m, 1H), 5.05 (s, 1H), 4.83 (s, 2H), 3.45 (s, 3H).

[1062] Example 20b

[1063] The first eluted enantiomer of 12 mg

[1064] Chiral SFC Rt = 2.31 min (Mobile phase: 5%-55% MeOH Column: (RR) Whelk-O1 4.6x100 mm 5um column, run for 6 min)

[1065] LCMS Rt = 1.91 min; MS m / z 388.0 [M+H]+; [Method 4]

[1066] 1 H NMR (400 MHz, DMSO-d6) δ 10.11 (d, J = 2.0 Hz, 1H), 7.33 - 6.80 (m, 4H), 5.77 - 5.65 (m, 1H), 5.64 - 5.51 (m, 1H), 5.05 (s, 1H), 4.83 (s, 2H), 3.45 (s, 3H).

[1067] Example 21: Methyl (R)-4-(2-cyclopropyl-5-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1068]

[1069] Step 1: 2-Cyclopropyl-5-fluorobenzaldehyde

[1070]

[1071] A degassed solution of 2-bromo-5-fluorobenzaldehyde (2.03 g, 10.00 mmol), cyclopropaneboronic acid (1.031 g, 12.00 mmol), and PdCl2(dppf)-CH2Cl2 adduct (0.817 g, 1.000 mmol) in dioxane (20 mL) and 2.0 M aqueous K2CO3 (10.00 mL, 20.00 mmol) was stirred and heated to 80 °C. After completion, the reaction mixture was cooled to room temperature and then partitioned between water and ethyl acetate. The organic phase was washed with water and brine, then dried over MgSO4 and concentrated. The crude was purified by flash chromatography on silica [0 - 20% ethyl acetate in heptane] to afford 800 mg of the title compound, 49% yield. The crude product was used without further purification.

[1072] Step 2: Methyl 4-(2-cyclopropyl-5-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1073]

[1074] According to General Procedure II, the title compound was prepared from ethyl 4-chloro-3-oxobutanoate (346 mg, 2.10 mmol), 2-cyclopropyl-5-fluorobenzaldehyde (345 mg, 2.10 mmol) prepared in Step 1, and methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 280 mg, 2.10 mmol). The racemic methyl 4-(2-cyclopropyl-5-fluorophenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate was separated into its enantiomers using chiral SFC (mobile phase: 25% methanol (containing 0.2% NH4OH / CO2), at 80 g / min; column: (SS)Whelk-O1 4.6 x 100 mm).

[1075] Example 21

[1076] 21.4 mg of the first eluting enantiomer as a white solid (5.6%).

[1077] Chiral SFC Rt = 2.41 min (mobile phase: 5% - 55% MeOH w / 10 mM NH4OH / CO2 5 mL / min 120 bar; column: (SS)Whelk-O1 4.6 x 100 mm 5 μm).

[1078] LCMS Rt = 0.95 min; MS m / z 362.5 [M+H]+; [Method 4].

[1079] 1 1H NMR (400 MHz, DMSO-d6) δ ppm 10.08 (s, 1H), 7.08 - 6.95 (m, 1H), 6.93 - 6.82 (m, 2H), 5.78 - 5.58 (m, 2H), 5.37 (s, 1H), 4.90 - 4.77 (m, 2H), 3.45 (s, 3H), 1.00 - 0.80 (m, 3H), 0.54 - 0.46 (m, 1H). The benzylic CH on the cyclopropyl is hidden under the DMSO peak.

[1080] Example 21b

[1081] 10.3 mg of the second eluting enantiomer as a white solid (2.7%).

[1082] Chiral SFC Rt = 2.69 min (mobile phase: 5% - 55% MeOH w / 10 mM NH4OH / CO2 5 mL / min 120 bar; column: (SS) Whelk-O1 4.6 x 100 mm 5 μm).

[1083] LCMS Rt = 0.95 min; MS m / z 362.5 [M+H]+; [Method 4].

[1084] 1 1H NMR (400 MHz, DMSO-d6) δ ppm 10.08 (s, 1H), 7.08 - 6.95 (m, 1H), 6.93 - 6.82 (m, 2H), 5.78 - 5.58 (m, 2H), 5.37 (s, 1H), 4.90 - 4.77 (m, 2H), 3.45 (s, 3H), 1.00 - 0.80 (m, 3H), 0.54 - 0.46 (m, 1H).

[1085] Example 22: Methyl (R)-4-(2-cyclopropylphenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1086]

[1087] Step 1: 2-Cyclopropylbenzaldehyde

[1088]

[1089] A degassed solution of 2-bromobenzaldehyde (1 g, 5.40 mmol), cyclopropaneboronic acid (0.557 g, 6.49 mmol), and PdCl2(dppf).CH2Cl2 adduct (0.441 g, 0.540 mmol) in dioxane (15 mL) and 2.0 M aqueous K2CO3 (5.40 mL, 10.81 mmol) was stirred and heated to 80 °C. After completion, the reaction mixture was cooled to room temperature and then partitioned between water and ethyl acetate. The organic phase was washed with water and brine, then dried over MgSO4 and concentrated. The crude product was purified by flash chromatography on silica [0 - 20% ethyl acetate in heptane] to afford 420 mg of the title compound, 53% yield. The crude product was used without further purification.

[1090] Step 2: Methyl 4-(2-cyclopropylphenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1091]

[1092] The title compound was prepared from ethyl 4-chloro-3-oxobutanoate (0.204 mL, 1.500 mmol), 2-cyclopropylbenzaldehyde (219 mg, 1.5 mmol) prepared in Step 1, and methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 200 mg, 1.50 mmol) according to General Procedure II. The racemic methyl-4-(2-cyclopropylphenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate was separated into its enantiomers using chiral SFC (mobile phase: 5% - 55% methanol (containing 10 mM NH4OH / CO2), at 3 mL / min; column: (SS)WHO; 1 4.6 x 100 mm).

[1093] Example 22

[1094] 7.9 mg of the first eluting enantiomer as a white solid (3.0%).

[1095] Chiral SFC Rt = 2.77 min

[1096] LCMS Rt = 0.93 min; MS m / z 344.5 [M+H]+; [Method 4].

[1097] 11H NMR (400 MHz, methanol-d4) δ ppm 6.91 - 7.23 (m, 4H), 5.73 (d, J = 1.96 Hz, 1H), 5.61 (d, J = 2.93 Hz, 1H), 5.48 (s, 1H), 4.82 (s, 2H), 3.50 (s, 3H), 2.53 - 2.65 (m, 1H), 0.86 - 0.99 (m, 3H), 0.58 (d, J = 5.87 Hz, 1H).

[1098] Example 22b

[1099] 7.5 mg of the second eluted enantiomer (2.9%) as a white solid.

[1100] Chiral SFC Rt = 3.19 min

[1101] LCMS Rt = 0.93 min; MS m / z 344.5 [M+H]+; [Method 4].

[1102] 1 1H NMR (400 MHz, methanol-d4) δ ppm 6.91 - 7.23 (m, 4H), 5.73 (d, J = 1.96 Hz, 1H), 5.61 (d, J = 2.93 Hz, 1H), 5.48 (s, 1H), 4.82 (s, 2H), 3.50 (s, 3H), 2.53 - 2.65 (m, 1H), 0.86 - 0.99 (m, 3H), 0.58 (d, J = 5.87 Hz, 1H).

[1103] Example 23: Methyl (R)-4-(5-fluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1104]

[1105] The title compound was prepared from ethyl 4-chloro-3-oxobutanoate (0.318 mL, 2.342 mmol), commercially available 5-fluoro-2-(trifluoromethyl)benzaldehyde (450 mg, 2.342 mmol), and methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 312 mg, 2.342 mmol) according to General Procedure II. The racemic methyl 4-(5-fluoro-2-(trifluoromethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate was separated into its enantiomers using chiral SFC (mobile phase: 15% methanol (containing 0.2% ammonium hydroxide / CO2) at 80 g / min; column: 2.0 x 25.0 cm ChromegaChiral CC4).

[1106] Example 23

[1107] 12.2 mg of the first eluted enantiomer as a white solid (2.7%).

[1108] Chiral SFC Rt = 1.29 min (column: 4.6 x 100 mm Chiralcel OZ-H; isocratic 15% methanol with 0.1% isopropylamine; 125 bar

[1109] LCMS Rt = 0.92 min; MS m / z 390.0 [M+H]+; [Method 4].

[1110] 1 1H NMR (400 MHz, DMSO-d6) δ ppm 10.16 (br s, 1H), 7.70 (dd, J = 8.80, 5.38 Hz, 1H), 7.20 - 7.30 (m, 2H), 5.72 (s, 1H), 5.60 (s, 1H), 5.20 (s, 1H), 4.74 - 4.91 (m, 2H), 3.38 (s, 3H).

[1111] Example 23b

[1112] 14.0 mg of the second eluted enantiomer as a white solid (3.0%).

[1113] Chiral SFC Rt = 1.74 min (column: 4.6 x 100 mm Chiralcel OZ-H; isocratic 15% methanol (containing 0.1% isopropylamine); 125 bar)

[1114] LCMS Rt = 0.92 min; MS m / z 390.0 [M+H]+; [Method 4].

[1115] 11H NMR (400 MHz, DMSO-d6) δ ppm 10.16 (br s, 1H), 7.70 (dd, J = 8.80, 5.38 Hz, 1H), 7.20 - 7.30 (m, 2H), 5.72 (s, 1H), 5.60 (s, 1H), 5.20 (s, 1H), 4.74 - 4.91 (m, 2H), 3.38 (s, 3H).

[1116] Example 24: Methyl (R)-2-(fluoromethyl)-5-oxo-4-(2-(trifluoromethyl)phenyl)-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1117]

[1118] According to General Procedure II, the title compound was prepared from ethyl 4-chloro-3-oxobutanoate (0.286 mL, 2.10 mmol), commercially available 2-trifluoromethylbenzaldehyde (366 mg, 2.10 mmol), and methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 280 mg, 2.10 mmol). The racemic methyl 2-(fluoromethyl)-5-oxo-4-(2-(trifluoromethyl)phenyl)-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate was separated into its enantiomers using chiral SFC (mobile phase: 20% methanol (containing 0.2% ammonium hydroxide) at 80 g / min; column: 2.0 x 25.0 cm ChromegaChiral CC4).

[1119] Example 24

[1120] 7.9 mg of the first eluted enantiomer (2.0%) as a white solid.

[1121] Chiral SFC Rt = 1.19 min (mobile phase: 20% methanol (containing 0.1% isopropylamine / CO2) at 4 mL / min; column: Chiralcel OZ-H; 1 4.6 x 100 mm).

[1122] LCMS Rt = 0.91 min; MS m / z 370.0 [M+H]+; [Method 4].

[1123] 11H NMR (400 MHz, DMSO-d6) δ ppm 10.12 (br s, 1H), 7.60 (d, J = 7.82 Hz, 2H), 7.49 (d, J = 7.83 Hz, 1H), 7.31 - 7.42 (m, 1H), 5.70 (s, 1H), 5.58 (s, 1H), 5.19 (s, 1H), 4.81 (s, 2H), 3.37 (s, 3H).

[1124] Example 24b

[1125] 9.3 mg of the second eluted enantiomer as a white solid (2.4%).

[1126] Chiral SFC Rt = 1.72 min (mobile phase: 20% methanol (containing 0.1% isopropylamine / CO2), at 4 mL / min; column: Chiralcel OZ-H; 1 4.6 x 100 mm).

[1127] LCMS Rt = 0.91 min; MS m / z 370.0 [M+H]+; [Method 4].

[1128] 1 1H NMR (400 MHz, DMSO-d6) δ ppm 10.12 (br s, 1H), 7.60 (d, J = 7.82 Hz, 2H), 7.49 (d, J = 7.83 Hz, 1H), 7.31 - 7.42 (m, 1H), 5.70 (s, 1H), 5.58 (s, 1H), 5.19 (s, 1H), 4.81 (s, 2H), 3.37 (s, 3H).

[1129] Example 25: Methyl (R)-4-(3-fluoro-2-(2,2,2-trifluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1130]

[1131] Step 1: 2-(2-Bromo-3-fluorophenyl)-1,3-dioxolane

[1132]

[1133] To a solution of 2-bromo-3-fluorobenzaldehyde (60 g, 295.56 mmol) and ethylene glycol (65.4 mL, 1182.2 mmol) in toluene (600 mL) was added p-toluenesulfonic acid monohydrate (28.11 g, 147.78 mmol). The resulting solution was stirred at 120 °C for 24 h using a Dean-Stark apparatus. The solvent was added to water (2 L) and extracted into EtOAc (3 L). The EtOAc was washed with saturated NaHCO3 solution (1 L), brine, and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to afford the title compound 2-(2-bromo-3-fluorophenyl)-1,3-dioxolane (60 g) as a colorless liquid.

[1134] 1 H NMR (400 MHz, CDCl3) δ 7.42 - 7.37 (m, 1H), 7.34 - 7.29 (m, 1H), 7.16 - 7.10 (m, 1H), 6.10 (s, 1H), 4.19 - 4.09 (m, 4H).

[1135] Step 2: 2-(1,3-dioxolan-2-yl)-6-fluorobenzaldehyde

[1136]

[1137] Under a nitrogen atmosphere, a solution of 2-(2-bromo-3-fluorophenyl)-1,3-dioxolane (from step 1, 7 g, 28.45 mmol) in THF (70 mL) was cooled to -78 °C. Then n-butyllithium (13.66 mL, 2.5 M, 58.5 mmol) in hexane solution was added dropwise over 10 min. The resulting mixture was stirred at -78 °C for 45 min, then DMF (2.5 g, 34.15 mmol) was added and the resulting mixture was stirred at -78 °C for 1.15 h. The reaction mixture was quenched with saturated ammonium chloride solution (50 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with water (50 mL), followed by brine (50 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→50% ethyl acetate in petroleum ether) to afford the title compound 2-(1,3-dioxolan-2-yl)-6-fluorobenzaldehyde (5 g) as a colorless liquid.

[1138] 11H NMR (300 MHz, chloroform-d) δ 10.52 (s, 1H), 7.67 - 7.53 (m, 2H), 7.22 - 7.12 (m, 1H), 6.50 (s, 1H), 4.27 - 3.99 (m, 4H).

[1139] Step 3: 2-(2-(2,2-Difluorovinyl)-3-fluorophenyl)-1,3-dioxolane

[1140]

[1141] Under a nitrogen atmosphere, triphenylphosphine (28.1 g, 107.1 mmol), potassium iodide (11.85 g, 71.4 mmol), and 2-(1,3-dioxolan-2-yl)-6-fluorobenzaldehyde (from Step 2, 7 g, 35.7 mmol) were added to a solution of acetonitrile (65 mL). The reaction was stirred at 70 °C for 30 min, and then methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (12 g, 62.4 mmol) was added slowly over a 10-min period (the color of the material turned yellow during addition). The resulting mixture was stirred at 70 °C for an additional 3 h, then cooled to room temperature, diluted with diethyl ether, and the precipitated solid was removed by filtration and washed with diethyl ether (100 mL). The resulting solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→5% ethyl acetate in petroleum ether) to give the title compound 2-(2-(2,2-difluorovinyl)-3-fluorophenyl)-1,3-dioxolane (6.9 g) as a pale yellow liquid.

[1142] 1 1H NMR (300 MHz, chloroform-d) δ 7.39 (dd, J = 7.8, 1.4 Hz, 1H), 7.31 (m, 1H), 7.11 (m, 1H), 5.87 (s, 1H), 5.39 (m, 1H), 4.21 - 3.95 (m, 4H).

[1143] Step 4: 2-(3-Fluoro-2-(2,2,2-trifluoroethyl)phenyl)-1,3-dioxolane

[1144]

[1145] 2-(2-(2,2-Difluorovinyl)-3-fluorophenyl)-1,3-dioxolane (from Step 3, 2.7 g, 11.73 mmol) and a solution of tetrabutylammonium fluoride in THF (13.7 g, 1 M, 13.7 mmol) were heated to 70 °C in a sealed system for 2 h. The reaction was allowed to cool, diluted with diethyl ether, and washed with water (50 mL), saturated NaHCO3 solution (100 mL), and brine and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to afford the title compound 2-(3-fluoro-2-(2,2,2-trifluoroethyl)phenyl)-1,3-dioxolane (1.2 g) as a colorless liquid.

[1146] 1 H NMR (300 MHz, chloroform-d) δ 7.49 - 7.42 (m, 1H), 7.35 (m, 1H), 7.17 - 7.05 (m, 1H), 6.03 (s, 1H), 4.25 - 3.90 (m, 4H), 3.75 (m, 2H).

[1147] Step 5: 3-Fluoro-2-(2,2,2-trifluoroethyl)benzaldehyde

[1148]

[1149] 6N HCl (5 mL) was added to a solution of 2-(3-fluoro-2-(2,2,2-trifluoroethyl)phenyl)-1,3-dioxolane (from Step 4, 2 g, 8.0 mmol) in diethyl ether (40 mL). The resulting solution was stirred at room temperature for 3 h. It was washed with water (50 mL), saturated NaHCO3 solution (100 mL), brine, and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→5% ethyl acetate in petroleum ether) to afford the title compound 3-fluoro-2-(2,2,2-trifluoroethyl)benzaldehyde (1.2 g) as a colorless liquid. The crude product was used in the next step without further purification.

[1150] Step 6: 3-Ethoxycyclopent-2-en-1-one

[1151]

[1152] At room temperature, pTSA (193 mg, 1.019 mmol) and EtOH (22.61 ml, 387.35 mmol) were added to a stirred solution of cyclopentane-1,3-dione (5.0 g, 50.96 mmol) in toluene (70 mL). The resulting mixture was stirred at 120 °C for 10 h using a Dean-Stark apparatus. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to afford the title compound 3-ethoxycyclopent-2-en-1-one as a brown solid (4.3 g).

[1153] 1 H NMR (400 MHz, CDCl3) δ 5.26 (s, 1H), 4.02 (q, J = 6.6 Hz, 2H), 2.60 - 2.55 (m, 2H), 2.44 - 2.40 (m, 2H), 1.39 (t, J = 7.2 Hz, 3H).

[1154] Step 7: 3-Aminocyclopent-2-en-1-one

[1155]

[1156] At room temperature, ammonium hydroxide solution (25 mL, 387.35 mmol) was added to a stirred solution of 3-ethoxycyclopent-2-en-1-one (from Step 6, 4.3 g, 34.08 mmol) in ethanol (50 mL). The resulting mixture was stirred at 85 °C for 16 h. The solvent was removed under reduced pressure to afford the title compound 3-aminocyclopent-2-en-1-one as a brown solid (3.2 g).

[1157] LCMS Rt = 0.114 min; MS m / z 98.2 [M+H]+; [Method 7]

[1158] Step 8: Methyl 4-(3-fluoro-2-(2,2,2-trifluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1159]

[1160] To a solution of aldehyde (from step 5, 200 mg, 0.9708 mmol), methyl 4-fluoro-3-oxobutanoate (method 2, step 1 of intermediate B, 130 mg, 0.9708 mmol) in EtOH (2 mL) was added 3-aminocyclopent-2-en-1-one (from step 7, 94 mg, 0.9708 mmol). The reaction mixture was stirred at 80 °C for 18 h. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on silica gel (0→70% ethyl acetate in petroleum ether) to afford the title compound as an off-white solid. The racemic mixture was separated into its enantiomers using chiral preparative HPLC [method 6].

[1161] Example 25

[1162] The second eluting enantiomer (25 mg) obtained as a white solid.

[1163] Chiral HPLC Rt = 7.610 min; [Chiral analytical method 4]

[1164] LCMS Rt = 1.533 min; MS m / z 401.75 [M+]+; [Method 12]

[1165] 1 1H NMR (400 MHz, CD3OD) δ 7.31 - 7.25 (m, 1H), 7.04 (d, J = 8 Hz, 1H), 6.97 - 6.90 (m, 1H), 5.76 (d, J = 3.2 Hz, 1H), 5.64 (d, J = 2.8 Hz, 2H), 5.08 (s, 1H), 4.79 - 4.60 (m, 2H), 3.70 - 3.55 (m, 2H), 3.44 (s, 3H), 2.66 - 2.61 (m, 2H).

[1166] Example 25b

[1167] The first eluting enantiomer (24 mg) obtained as a white solid.

[1168] Chiral HPLC Rt 7.103 min; [Chiral analytical method 4]

[1169] LCMS Rt = 1.533 min; MS m / z 401.75 [M+]+; [Method 12]

[1170] 11H NMR (400 MHz, CD3OD) δ 7.31 - 7.25 (m, 1H), 7.04 (d, J = 8 Hz, 1H), 6.97 - 6.90 (m, 1H), 5.76 (d, J = 3.2 Hz, 1H), 5.64 (d, J = 2.8 Hz, 2H), 5.08 (s, 1H), 4.79 - 4.60 (m, 2H), 3.70 - 3.55 (m, 2H), 3.44 (s, 3H), 2.66 - 2.61 (m, 2H).

[1171] Example 26: Methyl (R)-4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1172]

[1173] Step 1: 2-(2-(2,2-Difluoroethyl)-3-fluorophenyl)-1,3-dioxolane

[1174]

[1175] To a solution of 2-(2-(2,2-difluorovinyl)-3-fluorophenyl)-1,3-dioxolane (from Example 25, Step 3, 3 g, 13.04 mmol) in ethyl acetate (60 mL) was added 10% Pd-C (1 g). The resulting reaction mixture was kept in a Parr shaker at 60 psi pressure under a hydrogen atmosphere at room temperature for 48 h. The reaction mixture was filtered through a pad of Celite and washed with ethyl acetate (30 mL). The filtrate was concentrated under reduced pressure and the crude product was purified by flash chromatography on silica (0→30% ethyl acetate in petroleum ether) to give the title compound 2-(2-(2,2-difluoroethyl)-3-fluorophenyl)-1,3-dioxolane (3 g) as a colorless liquid.

[1176] 1 1H NMR (300 MHz, CDCl3) δ 7.42 - 7.35 (m, 1H), 7.34 - 7.27 (m, 1H), 7.15 - 7.08 (m, 1H), 6.31 - 5.78 (m, 2H), 4.19 - 4.02 (m, 4H), 3.38 (m, 2H).

[1177] Step 2: 2-(2,2-Difluoroethyl)-3-fluorobenzaldehyde

[1178]

[1179] To a solution of 2-(2-(2,2-difluoroethyl)-3-fluorophenyl)-1,3-dioxolane (from Step 1, 3 g, 12.93 mmol) in diethyl ether (40 mL) was added 6 N HCl (5 mL). The resulting solution was stirred at room temperature for 3 h. This was washed with water (50 mL), saturated NaHCO3 solution (100 mL) and brine, and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica (0→5% ethyl acetate in petroleum ether) to give the title compound 2-(2,2-difluoroethyl)-3-fluorobenzaldehyde (1.9 g) as a colorless liquid.

[1180] 1 1H NMR (400 MHz, CDCl3) δ 10.14 (d, J = 1.6 Hz, 1H), 7.70 - 7.63 (m, 1H), 7.52 (td, J = 8.0, 7.9, 5.2 Hz, 1H), 7.35 - 7.30 (m 1H), 6.03 (m, 1H), 3.83 - 3.58 (m, 2H).

[1181] Step 3: Methyl (R)-4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate and Methyl (S)-4-(2-(2,2-difluoroethyl)-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1182]

[1183] To a solution of the aldehyde (from Step 2, 200 mg, 1.063 mmol), methyl 4-fluoro-3-oxobutanoate (Method 2, Step 1 of Intermediate B, 142 mg, 1.063 mmol) in EtOH (2 mL) was added 3-aminocyclopent-2-en-1-one (Example 25, Step 7, 103 mg, 1.063 mmol). The reaction mixture was stirred at 80 °C for 18 h. The solvent was removed under reduced pressure to give the crude compound. The crude product was purified by flash chromatography on silica (0→70% ethyl acetate in petroleum ether) to give the title compound (100 mg) as an off-white solid. The racemic mixture was separated into its enantiomers using chiral preparative HPLC [Method 6].

[1184] Example 26

[1185] The first eluted enantiomer (25 mg) obtained as a white solid.

[1186] Chiral HPLC Rt 7.341 min; [Chiral analysis method 4]

[1187] LCMS Rt = 1.505 min; MS m / z 384.2 [M+1]+; [Method 12]

[1188] 1 H NMR (400 MHz, CD3OD) δ 7.25 - 7.17 (m, 1H), 7.01 (d, J = 8 Hz, 1H), 6.92 - 6.85 (m, 1H), 6.80 - 6.40 (m, 1H), 5.76 (d, J = 3.2 Hz, 1H), 5.64 (d, J = 2.8 Hz, 1H), 5.08 (s, 1H), 4.79 - 4.60 (m, 2H), 3.70 - 3.55 (m, 2H), 3.60 - 3.40 (m, 1H), 3.52 (s, 3H), 2.75 - 2.65 (m, 2H).

[1189] Example 26b

[1190] The second eluted enantiomer obtained as a white solid (26 mg).

[1191] Chiral HPLC Rt 9.044 min; [Chiral analysis method 4]

[1192] LCMS Rt = 1.505 min; MS m / z 384.2 [M+1]+; [Method 12]

[1193] 1 H NMR (400 MHz, CD3OD) δ 7.25 - 7.17 (m, 1H), 7.01 (d, J = 8 Hz, 1H), 6.92 - 6.85 (m, 1H), 6.80 - 6.40 (m, 1H), 5.76 (d, J = 3.2 Hz, 1H), 5.64 (d, J = 2.8 Hz, 1H), 5.08 (s, 1H), 4.79 - 4.60 (m, 2H), 3.70 - 3.55 (m, 2H), 3.60 - 3.40 (m, 1H), 3.52 (s, 3H), 2.75 - 2.65 (m, 2H).

[1194] Example 27: Methyl (R)-4-(3-fluoro-2-((R or S)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1195]

[1196] Step 1: 1-(2-(1,3-Dioxolan-2-yl)-6-fluorophenyl)ethan-1-ol

[1197]

[1198] At -78 °C under a nitrogen atmosphere, n-BuLi (58.3 mL, 2.5 M, 147.71 mmol) in a hexane solution was added dropwise over 10 min to a solution of 2-(2-bromo-3-fluorophenyl)-1,3-dioxolane (from Example 25, Step 1, 30 g, 121.42 mmol) in THF (300 mL). The resulting mixture was stirred at -78 °C for 1 h, then acetaldehyde (6.42 g, 145.71 mmol) was added, and the reaction was stirred at -78 °C for 1 h. The reaction mixture was quenched with saturated ammonium chloride solution (50 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with water (50 mL), washed with brine (50 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to give the title compound 1-(2-(1,3-dioxolan-2-yl)-6-fluorophenyl)ethan-1-ol (15 g) as a colorless liquid.

[1199] 1 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.0 Hz, 1H), 7.30 - 7.18 (m, 1H), 7.10 - 7.01 (m, 1H), 6.15 (s, 1H), 5.30 - 5.25 (m, 1H), 4.15 - 3.95 (m, 4H), 2.75 - 2.69 (m, 1H), 1.58 (dd, J = 6.6, 22.8 Hz, 3H).

[1200] Step - 2: 2-(3-Fluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane

[1201]

[1202] At -78 °C, diethylaminosulfur trifluoride (19.3 mL, 141.37 mmol) was added to a solution of 1-(2-(1,3-dioxolan-2-yl)-6-fluorophenyl)ethan-1-ol (from Step 1, 15 g, 70.68 mmol) in dichloromethane (150 mL). The resulting solution was allowed to warm to RT and stirred for 1 h. The reaction mixture was quenched with saturated ammonium chloride (20 mL) at 0 °C and diluted with ethyl acetate (100 mL). The organic layer was separated, washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→10% ethyl acetate in petroleum ether) to afford the title compound 2-(3-fluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane (8.5 g) as a colorless liquid.

[1203] 1 1H NMR (300 MHz, CDCl3) δ 7.38 (d, J = 7.5 Hz, 1H), 7.30 - 7.18 (m, 1H), 7.13 - 7.01 (m, 1H), 6.20 - 5.95 (m, 1H), 6.10 (s, 1H), 4.16 - 4.02 (m, 4H), 1.74 (dd, J = 7.2, 23.1 Hz, 3H).

[1204] Step - 3: 3-Fluoro-2-(1-fluoroethyl)benzaldehyde

[1205]

[1206] At room temperature, 4N HCl (85 mL) was added to a solution of 2-(3-fluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane (from Step 2, 8.5 g, 69.38 mmol) in diethyl ether (150 mL). The resulting solution was stirred at room temperature for 3 h. This was washed with water (100 mL), saturated NaHCO3 solution (200 mL) and brine, and dried over Na2SO4. The solvent was removed under reduced pressure to afford the title compound 3-fluoro-2-(1-fluoroethyl)benzaldehyde (6.5 g) as a colorless liquid. (Note: The aldehyde obtained is volatile in nature).

[1207] Step - 4: Methyl 4-(3-fluoro-2-((R or S)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1208]

[1209] To a solution of aldehyde (from Step 3, 1 g, 5.876 mmol), methyl 4-fluoro-3-oxobutanoate (Step 1 of Intermediate B in Method 2, 780 mg, 5.876 mmol) in EtOH (10 mL) was added 3-aminocyclopent-2-en-1-one (Step 7 of Example 25, 570 mg, 5.876 mmol). The reaction mixture was stirred at 80 °C for 18 h. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (0→70% ethyl acetate in petroleum ether) to give the title compound (220 mg) as an off-white solid, a mixture of diastereomers of methyl 4-(3-fluoro-2-(1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate).

[1210] The diastereomer mixture was separated into its two isomers using chiral preparative purification [Method 2].

[1211] Using chiral preparative purification [Method 5], the first peak of the separated diastereomers was separated into its enantiomeric peaks 1 and 2.

[1212] Example 27

[1213] The second eluted enantiomer (55 mg) obtained as a white solid

[1214] Chiral HPLC Rt = 5.729 min; [Chiral Analytical Method 2]

[1215] LCMS Rt = 2.25 min; MS m / z 364.1 [M-H]−; [Method 7]

[1216] 1 1H NMR (400 MHz, CDCl3) δ 7.25 - 7.15 (m, 2H), 6.96 - 6.75 (m, 2H), 6.52 - 6.30 (m, 1H), 5.79 (s, 1H), 5.67 (s, 1H), 5.05 (s, 1H), 3.59 (s, 3H), 2.80 - 2.60 (m, 2H), 2.50 - 2.35 (m, 2H), 2.01 (dd, J = 6.4, 23.2 Hz, 3H).

[1217] Example 27b

[1218] The first eluted enantiomer (55 mg) obtained as a white solid.

[1219] Chiral HPLC Rt = 5.678 min; [Chiral Analytical Method 2]

[1220] LCMS Rt = 2.25 min; MS m / z 364.1 [M-H]-; [Method 7]

[1221] 1 H NMR (400 MHz, CDCl3) δ 7.25 - 7.15 (m, 2H), 6.96 - 6.75 (m, 2H), 6.52 - 6.30 (m, 1H), 5.79 (s, 1H), 5.67 (s, 1H), 5.05 (s, 1H), 3.59 (s, 3H), 2.80 - 2.60 (m, 2H), 2.50 - 2.35 (m, 2H), 2.01 (dd, J = 6.4, 23.2 HZ, 3H).

[1222] Using chiral preparative purification [Method 10], the second peak of the separated diastereomers was separated into its enantiomeric peaks 3 and 4.

[1223] Example 27c

[1224] The third eluted enantiomer (10 mg) as a white solid.

[1225] Chiral HPLC Rt = 8.702 min; [Chiral analysis method 2]

[1226] LCMS Rt = 2.22 min; MS m / z 364.1 [M-H]-; [Method 7]

[1227] 1 H NMR (400 MHz, CDCl3) δ 7.25 - 7.15 (m, 2H), 6.96 - 6.75 (m, 2H), 6.80 - 6.60 (m, 1H), 5.79 (s, 1H), 5.69 (s, 1H), 5.03 (s, 1H), 3.54 (s, 3H), 2.75 - 2.58 (m, 2H), 2.50 - 2.45 (m, 2H), 1.83 (dd, J = 6.4, 22.4 HZ, 3H).

[1228] Example 27d

[1229] The fourth eluted enantiomer (10 mg) as a white solid.

[1230] Chiral HPLC Rt = 9.851 min; [Chiral analysis method 2]

[1231] LCMS Rt = 2.22 min; MS m / z 364.1 [M-H]-; [Method 7]

[1232] 11H NMR (400 MHz, CDCl3) δ 7.25 - 7.15 (m, 2H), 6.96 - 6.75 (m, 2H), 6.80 - 6.60 (m, 1H), 5.79 (s, 1H), 5.69 (s, 1H), 5.03 (s, 1H), 3.54 (s, 3H), 2.75 - 2.58 (m, 2H), 2.50 - 2.45 (m, 2H), 1.83 (dd, J = 6.4, 22.4 Hz, 3H).

[1233] Example 28: Methyl (R)-4-(2-cyclopropyl-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1234]

[1235] Step 1: 2-Cyclopropyl-3-fluorobenzaldehyde

[1236]

[1237] To a solution of 2-bromo-3-fluorobenzaldehyde (15 g, 73.88 mmol) and cyclopropylboronic acid (7.61 g, 88.66 mmol) in toluene (160 mL) was added 2N K2CO3 (25.5 mL, 182.16 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II).DCM (6.03 g, 7.38 mmol). The resulting solution was degassed with argon for 10 min and stirred at 100 °C for 4 h. The reaction mixture was filtered and washed with ethyl acetate (200 mL). The filtrate was added to water (500 mL) and the product was extracted into ethyl acetate (2 L). The EtOAc phase was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (0→1% ethyl acetate in petroleum ether) to give the title compound 2-cyclopropyl-3-fluorobenzaldehyde (11.2 g) as a colorless liquid.

[1238] 1 1H NMR (300 MHz, CDCl3) δ 10.70 (s, 1H), 7.63 (dd, J = 1.2, 7.8 Hz, 1H), 7.35 - 7.15 (m, 2H), 2.14 - 2.02 (m, 1H), 1.19 - 1.09 (m, 2H), 0.85 - 0.75 (m, 2H).

[1239] Step 2: Methyl 4-(2-cyclopropyl-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1240]

[1241] To a solution of aldehyde (from Step 1, 900 mg, 5.481 mmol), methyl (Z)-3-amino-4-fluorobut-2-enoate (Intermediate B, 537.76 mg, 5.481 mmol) in ethanol (5 mL) was added cyclopentane-1,3-dione (875.68161 mg, 5.481 mmol). The reaction mixture was stirred at 80 °C for 16 h. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (0→70% ethyl acetate in petroleum ether) to give the title compound, methyl 4-(2-cyclopropyl-3-fluorophenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate (130 mg) as an off-white solid. The racemic mixture was separated into its enantiomers using chiral preparative HPLC [Method 11].

[1242] Example 28

[1243] The first eluted enantiomer (46 mg) obtained as a white solid.

[1244] Chiral HPLC Rt = 10.37 min; [Chiral Analytical Method 3]

[1245] LCMS Rt = 1.47 min; MS m / z 360.2 [M+H]+; [Method 7]

[1246] 1 1H NMR (400 MHz, CDCl3) δ 7.10 - 7.01 (m, 1H), 6.95 - 6.90 (m, 1H), 6.78 - 6.71 (m, 1H), 5.68 (d, J = 0.9 Hz, 1H), 5.58 - 5.52 (m, 2H), 3.53 (s, 3H), 2.70 - 2.65 (m, 2H), 2.45 - 2.12 (m, 4H), 1.34 - 1.25 (m, 1H), 1.05 - 0.89 (m, 2H), 0.80 - 0.69 (m, 1H).

[1247] Example 28b

[1248] The second eluted enantiomer (48 mg) obtained as a white solid.

[1249] Chiral HPLC Rt = 15.216 min; [Chiral Analytical Method 3]

[1250] LCMS Rt = 1.47 min; MS m / z 360.2 [M+H]+; [Method 7]

[1251] 1 1H NMR (400 MHz, CDCl3) δ 7.10 - 7.01 (m, 1H), 6.95 - 6.90 (m, 1H), 6.78 - 6.71 (m, 1H), 5.68 (d, J = 0.9 Hz, 1H), 5.58 - 5.52 (m, 2H), 3.53 (s, 3H), 2.70 - 2.65 (m, 2H), 2.45 - 2.12 (m, 4H), 1.34 - 1.25 (m, 1H), 1.05 - 0.89 (m, 2H), 0.80 - 0.69 (m, 1H).

[1252] Example 29: Methyl (R)-4-(3,5-difluoro-2-((S or R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1253]

[1254] Step 1: 2-(1,3-Dioxolan-2-yl)-4,6-difluorobenzaldehyde

[1255]

[1256] To a solution of 2-(3,5-difluoro-2-vinylphenyl)-1,3-dioxolane (from Step 2 of Example 3, 5 g, 23.58 mmol) and ruthenium chloride·XH2O (490 mg, 2.35 mmol) in dichloromethane (50 mL) and water (10 mL) was added diacetoxyiodobenzene (11.4 g, 35.37 mmol). The resulting solution was stirred at 30 °C for 2 h. The reaction mixture was filtered and washed with ethyl acetate (200 mL). The filtrate was dissolved in water (200 mL) and extracted into ethyl acetate (500 mL), and dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel (0→10% ethyl acetate in petroleum ether) to give the title compound 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde (3 g) as a colorless liquid.

[1257] 1 1H NMR (300 MHz, CDCl3) δ 10.41 (s, 1H), 7.35 (d, J = 9.6 Hz, 1H), 6.95 - 6.85 (m, 1H), 6.53 (s, 1H), 4.08 (s, 4H).

[1258] Step 2: 1-(2-(1,3-Dioxolan-2-yl)-4,6-difluorophenyl)ethan-1-ol

[1259]

[1260] At 0 °C, methylmagnesium bromide (2.33 mL, 3 M in ether, 4.67 mmol) was added to a solution of 2-(1,3-dioxolan-2-yl)-4,6-difluorobenzaldehyde (from Step 1, 1.0 g, 4.67 mmol) in dry tetrahydrofuran (10 mL). The resulting solution was stirred at room temperature for 1 h. The reaction mixture was quenched with saturated ammonium chloride (10 mL) at 0 °C and diluted with ethyl acetate (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound 1-(2-(1,3-dioxolan-2-yl)-4,6-difluorophenyl)ethan-1-ol (950 mg) as a colorless liquid. The crude compound was carried on to the next step without further purification.

[1261] 1 1H NMR (300 MHz, CDCl3) δ 7.17 (dd, J = 2.4, 6.3 Hz, 1H), 6.89 - 6.72 (m, 1H), 6.17 (s, 1H), 5.31 (dd, J = 6.9, 13.5 Hz, 1H), 4.15 - 4.02 (m, 4H), 2.64 - 2.59 (m.1H), 1.58 (d, J = 9.0 Hz, 3H).

[1262] Step 3: 2-(3,5-Difluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane

[1263]

[1264] At -78 °C, diethylaminosulfur trifluoride (1.0 g, 6.13 mmol) was added to a solution of 1-(2-(1,3-dioxolan-2-yl)-4,6-difluorophenyl)ethan-1-ol (from Step 2, 950 mg, 4.13 mmol) in dichloromethane (10 mL). The resulting solution was stirred at room temperature for 1 h. The reaction mixture was quenched with saturated ammonium chloride (100 mL) at 0 °C and diluted with ethyl acetate (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound 2-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane (600 mg) as a thick yellow liquid. The crude compound was carried on to the next step without further purification.

[1265] 11H NMR (300 MHz, CDCl3) δ 7.20 (dd, J = 2.4, 6.3 Hz, 1H), 6.87 - 6.75 (m, 1H), 6.15 - 5.98 (m, 1H), 6.11 (s, 1H), 4.15 - 4.02 (m, 4H), 1.70 (dd, J = 6.6, 22.8 Hz, 3H).

[1266] Step 4: 3,5-Difluoro-2-(1-fluoroethyl)benzaldehyde

[1267]

[1268] At room temperature, 4N HCl (2 mL) was added to a solution of 2-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-1,3-dioxolane (from Step 3, 600 mg, 2.58 mmol) in diethyl ether (10 mL). The resulting solution was stirred at room temperature for 3 h. This was washed with water (50 mL) and saturated NaHCO3 solution (100 mL), and dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound 3,5-difluoro-2-(1-fluoroethyl)benzaldehyde (200 mg) as a colorless liquid. (Note: The aldehyde obtained is volatile in nature)

[1269] 1 1H NMR (300 MHz, CDCl3) δ 10.44 (d, J = 3.0 Hz, 1H), 7.20 (d, J = 9.3 Hz, 1H), 7.10 - 6.98 (m, 1H), 6.43 - 6.19 (m, 1H), 1.78 (dd, J = 7.2, 23.1 Hz, 3H).

[1270] Step 5: 3-Ethoxycyclopent-2-en-1-one

[1271]

[1272] At room temperature, pTSA (193 mg, 1.019 mmol) and EtOH (22.61 ml, 387.35 mmol) were added to a stirred solution of cyclopentane-1,3-dione (5.0 g, 50.96 mmol) in toluene (70 mL). The resulting mixture was stirred at 120 °C for 10 h using a Dean - Stark apparatus. The solvent was removed under reduced pressure to give a crude compound. The crude product was purified by flash chromatography on silica (0→50% ethyl acetate in petroleum ether) to give the title compound 3-ethoxycyclopent-2-en-1-one (4.3 g) as a brown solid.

[1273] 11H NMR (400 MHz, CDCl3) δ 5.26 (s, 1H), 4.02 (q, J = 6.6 Hz, 2H), 2.60 - 2.55 (m, 2H), 2.44 - 2.40 (m, 2H), 1.39 (t, J = 7.2 Hz, 3H).

[1274] Step 6: 3-Aminocyclopent-2-en-1-one

[1275]

[1276] At room temperature, ammonium hydroxide solution (25 mL, 387.35 mmol) was added to a stirred solution of 3-ethoxycyclopent-2-en-1-one (from Step 5, 4.3 g, 34.08 mmol) in ethanol (50 mL). The resulting mixture was stirred at 85 °C for 16 h. The solvent was removed under reduced pressure to give the title compound 3-aminocyclopent-2-en-1-one (3.2 g) as a brown solid.

[1277] LCMS RT = 0.114 min; MS m / z 98.2 [M+H]+; [Method 7]

[1278] Step 7: Methyl 4-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate

[1279]

[1280] To a solution of the aldehyde (from Step 4, 700 mg, 3.7204 mmol), methyl 4-fluoro-3-oxobutanoate (Step 1; Method 2 of Intermediate B, 598.73 mg, 4.4645 mmol) and 3-aminocyclopent-2-en-1-one (Step 6, 361.32 mg, 3.7204 mmol) in tert-butanol (15 mL). The reaction mixture was stirred at 80 °C for 48 h. The solvent was removed under reduced pressure to give a crude compound. The crude product was purified by flash chromatography on silica gel (0→50% ethyl acetate in petroleum ether) to give the title compound methyl 4-(3,5-difluoro-2-(1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-4,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carboxylate (580 mg) as a mixture of diastereomers and a first diastereomer (320 mg, peak-1) as an off-white solid. The mixture (580 mg) was further purified by preparative HPLC (Method 8) to give a second diastereomer (80 mg, peak-2) as an off-white solid.

[1281] Using preparative chiral HPLC [Method 11], Peak - 1: 160 mg and Peak - 2: 80 mg were further separated into their enantiomers to obtain the four isomers of Example 29.

[1282] Example 29

[1283] The first eluted enantiomer (50 mg) as a white solid obtained using chiral HPLC [Method 11].

[1284] LCMS RT = 1.445 min; MS m / z 382.1 [M - H] - ; [Method 7]

[1285] This was further purified by preparative HPLC purification [Method 9], giving an off - white solid (25 mg).

[1286] Chiral HPLC: RT: 8.905 min; [Chiral Analytical Method 3]

[1287] LCMS RT = 1.445 min; MS m / z 382.1 [M - H] - ; [Method 13]

[1288] 1 1H NMR (400 MHz, DMSO - d6) δ 10.10 (s, 1H), 7.06 (ddd, J = 11.6, 8.8, 2.6 Hz, 1H), 6.82 - 6.68 (m, 1H), 6.36 (dq, J = 45.3, 6.5 Hz, 1H), 5.75 - 5.64 (m, 1H), 5.63 - 5.50 (m, 1H), 4.97 (s, 1H), 3.45 (s, 3H), 2.70 - 2.59 (m, 2H), 2.25 - 2.09 (m, 2H), 1.84 (dd, J = 23.0, 6.5 Hz, 3H).

[1289] Example 29b

[1290] The second eluted enantiomer (55 mg) as a white solid obtained using chiral HPLC [Method 11].

[1291] Chiral HPLC: RT: 10.197 min; [Chiral Analytical Method 3]

[1292] LCMS RT = 1.510 min; MS m / z 382.1 [M - H] - ; [Method 13]

[1293] 11H NMR (400 MHz, DMSO-d6) δ 10.10 (s, 1H), 7.06 (ddd, J = 11.6, 8.8, 2.6 Hz, 1H), 6.82 - 6.67 (m, 1H), 6.36 (dq, J = 45.4, 6.6 Hz, 1H), 5.75 - 5.64 (m, 1H), 5.63 - 5.48 (m, 1H), 4.97 (s, 1H), 3.45 (s, 3H), 2.71 - 2.59 (m, 2H), 2.27 - 2.16 (m, 2H), 1.84 (dd, J = 23.0, 6.5 Hz, 3H).

[1294] Example 29c

[1295] The third eluted enantiomer, 24.2 mg as a white solid, obtained using chiral HPLC [Method 11].

[1296] Chiral HPLC: RT: 14.455 min; [Chiral analytical method 3]

[1297] LCMS RT = 1.504 min; MS m / z 382.0 [M-H]-; [Method 10]

[1298] 1 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 7.14 - 6.96 (m, 1H), 6.75 (d, J = 9.8 Hz, 1H), 6.63 (dq, J = 44.2, 6.8 Hz, 1H), 5.71 (s, 1H), 5.59 (s, 1H), 4.88 (s, 1H), 3.47 (s, 3H), 2.62 (q, J = 4.5 Hz, 2H), 2.25 (t, J = 4.9 Hz, 2H), 1.68 (dd, J = 22.8, 6.5 Hz, 3H).

[1299] Example 29d

[1300] The fourth eluted enantiomer, 32.4 mg as a white solid, obtained using chiral HPLC [Method 11].

[1301] Chiral HPLC: RT: 18.561 min [Chiral analytical method 3]

[1302] LCMS RT = 1.514 min; MS m / z 382.0 [M-H]-; [Method 10]

[1303] 11H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 7.05 - 7.00 (m, 1H), 6.75 (d, J = 9.7 Hz, 1H), 6.62 (dt, J = 44.2, 6.6 Hz, 1H), 5.71 (s, 1H), 5.59 (s, 1H), 4.88 (s, 1H), 3.47 (s, 3H), 2.62 (q, J = 4.6 Hz, 2H), 2.25 (t, J = 4.9 Hz, 2H), 1.68 (dd, J = 22.8, 6.4 Hz, 3H).

[1304] Example 30: Methyl (R)-4-(2-ethylphenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1305]

[1306] Step 1: Methyl 4-(2-ethylphenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1307]

[1308] The title compound was synthesized using General Method I (using 2-ethylbenzaldehyde, 0.2 g, 1.44 mmol). The crude product was purified by silica column chromatography (2% → 3% methanol in DCM) to give the title compound, methyl 4-(2-ethylphenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate as a cream solid (0.165 g, 43%). LCMS Rt = 1.506 min; MS m / z 331.85 [M+H]+; [Method 10]

[1309] The racemic sample was separated into its enantiomers by chiral HPLC (column: LUX cellulose-4 (250 MM X 21.2 MM X 5 micron); mobile phase: hexane (A) EtOH:MeOH, 1:1 (B), flow rate: 15 mL).

[1310] Example 30

[1311] The first eluted enantiomer (30 mg) as a pale yellow solid.

[1312] Chiral HPLC Rt = 5.952 min (column: LUX cellulose-4 (150X4.6 mmX5μ); mobile phase: A = hexane, B: ethanol:methanol (50:50); flow rate: 1.0 mL / min).

[1313] LCMS Rt = 1.53 min; MS m / z 332.0 [M+H]+; [Method 7]

[1314] 1 H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 7.13 - 7.10 (m, 4H), 5.71 (s, 1H), 5.59 (s, 1H), 4.98 (s, 1H), 4.82 (s, 2H), 3.44 (s, 3H), 2.98 - 2.92 (m, 2H), 1.25 (t, J = 7.8 Hz, 3H).

[1315] Example 30b

[1316] The second eluted enantiomer as a pale yellow solid (29 mg).

[1317] Chiral HPLC Rt = 7.581 min (Column: LUX cellulose-4 (150X4.6 mmX5μ); Mobile phase: A = n-hexane, B: ethanol: methanol (50:50); Flow rate: 1.0 ml / min).

[1318] LCMS Rt = 1.528 min; MS m / z 332.0 [M+H]+; [Method 7]

[1319] 1 H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 7.13 - 7.10 (m, 4H), 5.71 (s, 1H), 5.59 (s, 1H), 4.98 (s, 1H), 4.82 (s, 2H), 3.44 (s, 3H), 2.98 - 2.92 (m, 2H), 1.25 (t, J = 7.8 Hz, 3H).

[1320] Example 31: Methyl (R)-2-(difluoromethyl)-4-(3-fluoro-2-((R or S)-1-fluoroethyl)phenyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1321]

[1322] Step 1: 5-Ethyl 3-methyl 2-(dimethoxymethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate

[1323]

[1324] A stirred solution of methyl 4,4-dimethoxy-3-oxobutanoate (2.0 g, 11.35 mmol), 3-fluoro-2-(1-fluoroethyl)benzaldehyde (from Example 27, Step 3, 1.93 g, 11.35 mmol), and ethyl (Z)-3-aminobut-2-enoate (1.46 g, 11.35 mmol) in ethanol (20 mL) was heated to 90 °C for 16 h. The reaction mixture was concentrated under reduced pressure to give the crude compound as a brown liquid. The crude compound was purified by flash chromatography on silica (9% → 10% ethyl acetate in hexanes) to give the title compound as a yellow viscous liquid. 5-Ethyl 3-methyl 2-(dimethoxymethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate (1.7 g, 34%).

[1325] The compound was used as a crude product without further analysis

[1326] Step 2: 3-Ethyl 5-methyl 4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-formyl-2-methyl-1,4-dihydropyridine-3,5-dicarboxylate

[1327]

[1328] A stirred solution of 5-ethyl 3-methyl 2-(dimethoxymethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate (from Step 1, 1.7 g, 3.86 mmol) in 1,4-dioxane (10.0 mL) was cooled to 0 °C. At 0 °C, 6N aqueous hydrochloric acid (15.0 mL) was added, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water, and the product was extracted into ethyl acetate. The combined organic layers were washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude compound was purified by flash chromatography on silica (12% → 15% ethyl acetate in hexanes) to give diastereomer 1 (400 mg, 26%) and diastereomer 2 (300 mg, 20%) of the title compound as yellow viscous liquids.

[1329] Diastereomer 1

[1330] LCMS Rt = 1.606 min; MS m / z 392.0 [M-H]−; [Method 10]

[1331] 11H NMR (300 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.05 (s, 1H), 7.33 (dd, J = 13.8, 7.2 Hz, 1H), 7.10 - 7.02 (m, 2H), 6.45 - 6.23 (m, 1H), 5.19 (s, 1H), 4.06 - 3.98 (m, 2H), 3.63 (s, 3H), 2.35 (s, 3H), 1.76 (dd, J = 22.8, 6.6 Hz, 3H), 1.13 (t, J = 6.9 Hz, 3H)

[1332] Diastereomer 2

[1333] LCMS Rt = 1.461 min; MS m / z 392.0 [M-H]−; [Method 14]

[1334] 1 1H NMR (300 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.07 (s, 1H), 7.33 (dd, J = 13.8, 7.2 Hz, 1H), 7.10 - 7.03 (m, 2H), 6.44 - 6.22 (m, 1H), 5.18 (s, 1H), 4.13 - 3.87 (m, 2H), 3.67 (s, 3H), 2.35 (s, 3H), 1.74 (dd, J = 22.8, 6.6 Hz, 3H), 1.11 (t, J = 6.9 Hz, 3H).

[1335] Step 3: 5-Ethyl 3-methyl 2-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate

[1336]

[1337] A stirred solution of 3-ethyl 5-methyl 4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-formyl-2-methyl-1,4-dihydropyridine-3,5-dicarboxylate (diastereomer 1 from step 2, 0.40 g, 1.01 mmol) in dichloromethane (5.0 mL) was cooled to -78 °C. At -78 °C, diethylaminosulfur trifluoride (0.16 mL, 1.22 mmol) was added in portions, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with ice-cold water, and the product was extracted into dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to afford the title compound as a brown viscous liquid. 5-Ethyl 3-methyl 2-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate (0.3 g).

[1338] LCMS Rt = 1.632 min; MS m / z 414.0 [M-H]-; [Method 10]

[1339] 1 H NMR (300 MHz, DMSO-d6) δ 9.38 (s, 1H), 7.58 - 7.23 (m, 2H), 7.09 - 7.01 (m, 2H), 6.43 - 6.22 (m, 1H), 5.12 (s, 1H), 4.08 - 3.97 (m, 2H), 3.58 (s, 3H), 2.34 (s, 3H), 1.75 (dd, J = 23.1, 6.0 Hz, 3H), 1.13 (t, J = 6.9 Hz, 3H)

[1340] Step 4: 3-Ethyl 5-methyl 2-(bromomethyl)-6-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate

[1341]

[1342] A solution of 5-ethyl 3-methyl 2-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate (from Step 3, 0.3 g, 0.722 mmol) in dichloromethane (5.0 mL) was cooled to -78 °C. At -78 °C, pyridinium tribromide (0.254 g, 0.794 mmol) was added and the mixture was stirred at the same temperature for 1 h. The reaction mixture was then stirred at room temperature for 30 min. The reaction mixture was diluted with ice-cold water and the product was extracted into dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude compound was purified by flash chromatography on silica gel (10%→12% ethyl acetate in hexane) to afford the title compound as a yellow viscous liquid. 3-Ethyl 5-methyl 2-(bromomethyl)-6-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate (0.2 g, 56%)

[1343] LCMS Rt = 1.653 min; MS m / z 491.95 [M-H] -; [Method 10]

[1344] 1 H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 7.40 - 7.34 (m, 2H), 7.11 - 7.02 (m, 2H), 6.40 - 6.24 (m, 1H), 5.16 (s, 1H), 4.73 (dd, J = 34, 9.2 Hz, 2H), 4.14 - 4.05 (m, 2H), 3.58 (s, 3H), 1.75 (dd, J = 22.4, 6.4 Hz, 3H), 1.18 (t, J = 7.4 Hz, 3H)

[1345] Step 5: Methyl 2-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate

[1346]

[1347] At room temperature, potassium hydroxide (0.005 g, 0.101 mmol) was added to a solution of 3-ethyl 5-methyl 2-(bromomethyl)-6-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate (from Step 4, 0.1 g, 0.202 mmol) in chloroform (5.0 mL). The reaction mixture was heated to 60 °C and stirred for 8 h. At room temperature, tetra-n-butylammonium iodide (0.007 g, 0.020 mmol) was added and the reaction mixture was heated to 65 °C for 12 h. The reaction mixture was diluted with water and the product was extracted into dichloromethane. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: ZORBAX XDB (250 mm x 21.2 mm), 5.0 μ; mobile phase: A = water B = ACN; flow rate: 18 ml / min) to give the title compound (methyl 2-(difluoromethyl)-4-(3-fluoro-2-(1-fluoroethyl)phenyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate) as a cream solid (20 mg, 26%).

[1348] LCMS Rt = 1.503 min; MS m / z 384.05 [M-H]-; [Method 10]

[1349] The racemic sample was separated into its enantiomers by chiral HPLC (column: CHIRALPAK IJ, 250 MM X 4.3 MM X 5 micron; mobile phase: A = hexane, B = 0.1% HCOOH:EtOH in MeOH, 1:1, flow rate: 5 mL).

[1350] Example 31:

[1351] The first elution peak (3.4 mg) of chiral HPLC as a cream solid.

[1352] Chiral HPLC Rt = 6.393 min (column: CHIRAL PAK IJ (250X 4.6 mm X 5μ); mobile phase: A: n-hexane B: ethanol; flow rate: 1.0 mL / min.

[1353] LCMS Rt = 1.502 min; MS m / z 384.05 [M-H]- [Method 10]

[1354] 11H NMR (300 MHz, DMSO-d6) δ 10.43 (s, 1H), 7.64 - 7.28 (m, 2H), 7.10 - 7.02 (m, 2H), 6.44 - 6.22 (m, 1H), 5.13 (s, 1H), 4.88 (s, 2H), 3.49 (s, 3H), 1.80 (dd, J = 22.8, 6.0 Hz, 3H)

[1355] Example 31b

[1356] The second elution peak (3 mg) of chiral HPLC as a cream solid.

[1357] Chiral HPLC Rt = 7.605 min (column: CHIRAL PAK IJ (250X 4.6 mm X 5 μ); mobile phase: A: n-hexane B: ethanol; flow rate: 1.0 mL / min.

[1358] LCMS Rt = 1.500 min; MS m / z 384.05 [M-H]-. [Method 10]

[1359] 1 1H NMR (300 MHz, DMSO-d6) δ 10.42 (s, 1H), 7.59 - 7.32 (m, 2H), 7.10 - 7.02 (m, 2H), 6.42 - 6.25 (m, 1H), 5.13 (s, 1H), 4.88 (dd, J = 19.2, 16.4 Hz, 2H), 3.49 (s, 3H), 1.80 (dd, J = 23.2, 6.4 Hz, 3H).

[1360] Crystalline form

[1361] X-ray powder diffraction measurement (XRPD)

[1362] XRPD was performed using a Bruker D8 Advance diffractometer equipped with a nickel filter monochromator and a LYNXEYE (1D mode) detector with an opening angle: 2.948°, using Cu-Kα 1,2Radiation (wavelength 0.15419 nm). The diffraction pattern was recorded at a tube voltage of 40 kV and a tube current of 40 mA. In the angular range of 2° to 40° 2-θ under ambient conditions, a step size of 0.0164° (2-θ value) per step of 0.3 seconds was applied. The scanning time was 768 seconds. The typical accuracy of the 2-θ value was in the range of ±0.2° 2-θ, preferably ±0.1° 2-θ. Thus, for example, under standard conditions on most X-ray diffractometers, the diffraction peak of crystalline Form A of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate that appears at 10.4° 2-θ could appear in the range from (10.4 - 0.2)° to (10.4 + 0.2)° 2-θ, preferably from (10.4 - 0.1)° to (10.4 + 0.1)° 2-θ.

[1363] Crystalline Form A

[1364] At room temperature, crude methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (130 g, 0.35 mol) was slurried in diethyl ether (520 ml) for 3 hours to obtain a suspension, and the suspension was filtered. Then the wet cake was dried under vacuum at 60 °C for 72 hours to provide methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (Form A) (100 g, 0.27 mol) with a yield of 77%.

[1365] Crystalline Form B

[1366] Crystalline Form B was prepared by dissolving 100 mg of Form A in sufficient isopropyl acetate to obtain a solution. Then sufficient heptane was added at 55 °C until a turbid suspension was obtained. The suspension was maintained at 55 °C for a certain time, then cooled to 25 °C and equilibrated at 25 °C for 2 to 4 hours. The solid portion was separated and analyzed by XRPD to confirm the presence of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (Form B).

[1367] Alternative synthesis: 400 mL of heptane was added to a 500 mL reactor and heated to 55 °C. At room temperature, 20 g of Form A was added to 60 mL of isopropyl acetate. At 55 °C, approximately 2 g of Form B was added as a seed to the reactor and the resulting mixture was equilibrated at 55 °C for 30 min. Then, the isopropyl acetate solution of Form A in free form was added dropwise to the reactor over 2 h using an injection pump. The resulting mixture was equilibrated at 55 °C for 2 h. Then the mixture was cooled to 25 °C over 6 h and then equilibrated at 25 °C for an additional 10 h. Then the suspension was filtered and the wet cake was dried under vacuum at 50 °C for 4 h. Approximately 21 g of the final product was obtained, which was confirmed by XRPD to be in accordance with Form B. The water content measured by KF was 0.3%.

[1368] Crystalline Form C

[1369] Approximately 100 mg of methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate was crystallized with various stoichiometries of citric acid (base:acid ratios of 4:1, 3:1, 2:1 or 1:1) in tert-butyl methyl ether. The solid forms obtained were in accordance with methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (Form C).

[1370] Alternative synthesis: At 25 °C, 40 mL of heptane was added to a 100 mL EasyMax reactor. At room temperature, 2 g of Form A was dissolved in 5 mL of isopropyl acetate. Then, at 25 °C, 20 mg of a seed of Form C was added to the reactor. The resulting mixture was equilibrated at 25 °C for 30 min. The isopropyl acetate solution of Form A in free form was added dropwise to the reactor over a 2 h period using an injection pump. The resulting mixture was equilibrated at 25 °C for 2 h. Process control showed a mixture of Form C and Hydrate A. Then the solid material was slurried in 10 mL of isopropyl acetate / heptane (1 / 7) for 2 days. Then the suspension was filtered and dried at 40 °C for 18 h. The XRPD of the final product was in accordance with Form C. The water content measured by KF was 0.3%.

[1371] Crystalline Form Hydrate A

[1372] 1.97 g of Form A was weighed into a 100 mL EasyMax glass vial and dispersed in 35 mL of water. The suspension was stirred at 300 rpm for 3 hours at 50 °C by top stirring, then cooled to 25 °C at about 0.07 °C / min over 6 hours, and then kept stirring at 25 °C for 24 hours. The solid material was separated by filtration. The XRPD of the solid form was consistent with hydrate A.

[1373] Alternative synthesis: For example, hydrate A was obtained by evaporative crystallization of a clear saturated solution of crystalline Form B in acetone, acetonitrile / water (99.5:0.5), ethyl acetate or water. The mass loss shown by hydrate A by TGA was about 4.1%, which is close to that of the monohydrate (calculated water content of 4.7%). Hydrate A is slightly hygroscopic. The water uptake between 10% and 95% RH at 25 °C was about 0.2%.

[1374] Crystalline form hydrate B

[1375] 2 g of Form A was weighed into a 100 mL EasyMax glass vial and dispersed in 30 mL of methanol. The suspension was stirred at 300 rpm for 20 hours at 25 °C by top stirring. The solid material was separated by filtration, and the solid was exposed to 92% RH for 1 day. As confirmed by XRPD, the solid material was consistent with hydrate B.

[1376] Alternative synthesis: Hydrate B was obtained by air-drying crystalline Form B in the methanol solvate. The mass loss shown by hydrate B by TGA was about 4.4%, which is also close to that of the monohydrate (calculated water content of 4.7%). Hydrate B starts to dehydrate at below 20% RH and loses about 4.4% of its water by 0% RH. When the relative humidity increases subsequently, it only absorbs about 3% of water at 25 °C.

[1377] Differential scanning calorimetry

[1378] Differential scanning calorimetry was performed using a TA Discovery DSC in the temperature range of 30 °C to 300 °C at a scan rate of 10 °C / min under nitrogen with a flow rate of 25 mL / min. The sample mass used was about 2 mg.

[1379] Biological data

[1380] Many known calcium channel activators show activation of Ca V 1.2 through complex mechanisms. These molecules not only increase the peak current but also have additional mechanisms to increase the intracellular calcium concentration, for example, by shifting the voltage sensitivity of the channel towards more negative membrane potentials. Figure 6By depicting simulated cardiac action potentials from the epicardial environment and showing the effect of the shift of the voltage activated at 1.2 towards a more negative membrane potential to demonstrate these additional mechanisms. These additional mechanisms may drive or promote cardiovascular effects such as elevated blood pressure, changes in heart rate or contractility, and / or arrhythmias due to QT prolongation. For example, the O’Hara-Rudy model is used to study the effect of Ca V 1.2 regulation on action potential duration and the propensity for arrhythmias. It has been determined that a hyperpolarizing shift of >12 mV in the activation curve may potentially lead to a >15% QT prolongation and an increased risk of arrhythmias. Therefore, minimizing any shift in voltage sensitivity may lead to a reduced risk of QT prolongation and arrhythmias for a compound. V Compounds having the formula (I) are potent Ca

[1381] 1.2 activators that have biophysical characteristics that minimize the above-mentioned cardiovascular risks. First, compounds having the formula (I) limit their effect on voltage sensitivity by minimizing the hyperpolarizing shift to <9 mV, thereby reducing the likelihood of arrhythmias. Second, compounds having the formula (I) increase the Ca V 1.2 peak current by no more than 2.5-fold, thereby limiting the overactivation of the channel. Third, compounds having the formula (I) do not delay the inactivation of the Ca V 1.2 channel (which is a potential pathophysiological mechanism for the cardiac symptoms of Timothy syndrome). In addition, compounds having the formula (I) are designed to maximize brain exposure by not showing significant efflux in the brain. V Generation and maintenance of the Ca

[1382] Ca V 1.2-HEK293(AUX) cell line

[1383] The monoclonal Ca V 1.2-HEK293(AUX) cell line constitutively expresses human Ca V1.2 α1C (alpha1C) subunit (CACNA1C) and doxycycline-inducible expression of α2Δ (alpha2delta, α2Δ2) auxiliary subunit (CACNA2D2) and β2 (beta2) auxiliary subunit (CACNB2). To generate the cell line, expression vectors pcDNA5.0 / FRT-TO-CACNA2D2-FCS-P2A-CACNB2 and pCMV6-entry-CACNA1C were established by gene synthesis and cloning. Here, the pcDNA5.0 / FRT-TO plasmid was from Invitrogen, the pCMV6-entry was from Origene, FCS represents the furin protease cleavage site, P2A is a peptide self-cleavage sequence derived from porcine teschovirus-1, and FRT is the flippase recognition target site. Next, the parental line Flp-In TM 293T-Rex (Invitrogen) was transfected with pcDNA5.0 / FRT-TO-CACNA2D2-FCS-P2A-CACNB2 and the flippase vector pOG44 (Invitrogen) to establish the targeted integration of the CACNA2D2-FCS-P2A-CACNB2 expression cassette into the pre-engineered FRT site in Flp-In TM 293 T-Rex. Then the intermediate cell line was transfected with pCMV6-entry-CACNA1C to establish stable CACNA1C expression. Clonal isolation was achieved under neomycin selection. Cell clones (2-19B) with good voltage-dependent barium current (see electrophysiology methods below) were selected for the characterization of Ca V 1.2 activators.

[1384] To maintain the cell line, the cells were passaged twice a week. At each passage, the growth medium (Table 6) was completely removed, and the cells were rinsed successively with 10 ml of D-PBS and 5 ml of warm TrypLE TM Express enzyme (Gibco). The D-PBS and TrypLE TM Express enzyme were removed immediately after rinsing. Then the plate was left at room temperature for 3-5 minutes. Next, 10 mL of warm 37 °C complete medium was added to rinse the cell growth surface and collect the dissociated cells. The cells were counted and seeded into a new flask at a target density of 2-3×10 6 cells / T175 cm 2 flask.

[1385] Table 6. Growth medium for HEK293-Ca V 1.2 (AUX) cells

[1386] Reagent Concentration DMEM Heat-inactivated fetal bovine serum 10％ Hygromycin B 100 ug / ml 30 Blasticidin 10 ug / ml Geneticin (G418) 200 ug / mL

[1387] Using Ca V 1.2-HEK293(AUX) cell line and QPatch for electrophysiological characterization of Ca V 1.2 activators

[1388] Twenty-four hours before the electrophysiology experiment, doxycycline (1 μg / ml) was added to the growth medium (Table 6), and 25 μM verapamil was co-applied to prevent cell death caused by calcium influx. Just before the experiment, cell confluence should reach 70%-80%.

[1389] To harvest cells (from a T175 cm 2 flask as an example), the growth medium was completely removed, and the cells were rinsed with 10 mL of D-PBS. The D-PBS was aspirated and 10 mL of Detachin (Genlantis) was added, and the plate was placed in a 37°C incubator for 10 minutes. The detached cells were placed in a 15 mL conical tube and spun at 1000 rpm for 2 minutes. The supernatant was removed, and the cells were resuspended in QPatch complete medium (Table 7) to achieve the desired cell density of 1.5-3 million cells per QPatch run. 1.5 mL of cells was used for each experimental run.

[1390] The cell suspension was taken to the Sophion QPatch platform, which uses whole-cell voltage clamp to measure barium currents conducted through Ca V 1.2 on single-hole QPlates. The extracellular and intracellular patch clamp solutions are described in Tables 7 and 8 respectively. A dose-response assay protocol was used to determine the peak inward current (Emax) and potency (EC 50 ) of each compound. The protocol has eight liquid periods. The first liquid period is the stable current amplitude, which is monitored using repeated 200-ms voltage pulses stepping from -80 mV to 0 mV. The second liquid period is to determine the baseline current amplitude using a single 20-ms voltage pulse stepping from -80 mV to 0 mV in the presence of a vehicle control. The third to eighth liquid periods also use a single 20-ms voltage pulse stepping from -80 mV to 0 mV to determine the 6-dose response to compound treatment. The following equation was used to generate EC 50 : I 浓度 = I 基线 + (I 完整 - I 基线 ) * c n / (XC 50 n+c n ), where c is the concentration and n is the Hill coefficient constant. I 完整 is the maximum current achievable, and I 基线 is 0. The channel biophysics assay protocol is used to determine channel gating properties, including the current-voltage relationship (IV curve), the half-way channel activation voltage (V 1 / 2 ), the channel inactivation rate (tau), and the tail current amplitude. Among them, V 1 / 2 is obtained from the fitting equation: G(V) = G Vmin +(G Vmax - G Vmin ) / (1 + exp(-(V - V1 / 2) / V 斜率 ), where G represents conductance, G Vmin equals 0, G Vmax is the maximum conductance, and V slope is the slope factor. G(V) is pre-calculated from the equation G(V) = I(V) / (V - 0.06). For each depolarizing potential (V) applied in the experiment and the corresponding current amplitude (I(V)), 0.06 in the equation is the experimentally determined reversal potential in volts. This protocol has four liquid cycles. The first liquid cycle is the stable current amplitude, which is monitored using repeated 200-ms voltage pulses stepping from -80 mV to 0 mV. After the current amplitude stabilizes, the baseline value of the inactivation tau is determined by performing a single exponential fit on the inactivation phase of the current trace. The second liquid cycle is to measure the baseline values constituting the current-voltage relationship in the presence of the vehicle control, and the third liquid cycle is to measure the effect of the compound on the current-voltage relationship. During each of these two liquid cycles, the cells are given ten 20-ms voltage pulses, each pulse stepping from -80 mV to an increment value that ranges from -55 mV to +35 mV (increment size is 10 mV). During the fourth liquid cycle, 200-ms voltage pulses from -80 mV to 0 mV are delivered again to measure the effect of the compound on the inactivation tau.

[1391] Table 6. QPatch complete medium:

[1392] Reagent Concentration CHO - Serum-free medium (SFM) 1M HEPES 25 mM

[1393] Table 7. Extracellular solution for QPatch experiments

[1394]

[1395] Table 8. Intracellular solution for QPatch experiments. This solution is a mixture of 80% of the second part (stored at -80 °C) and 20% of the first part:

[1396] Part I:

[1397]

[1398] Part II:

[1399]

[1400] Evaluating the compound exposure-cFos induction (PK-PD) relationship in wild-type mice

[1401] Animal maintenance and ethics. All animals were housed under a regulated temperature and light cycle (22 °C, 12-hour light / 12-hour dark cycle) with unrestricted access to food and water. All animal experiments were conducted in accordance with the institutional guidelines for laboratory animal care and use approved by the Institutional Animal Care and Use Committee (IACUC) of Novartis Institutes for BioMedical Research, Inc. (Cambridge, Massachusetts, USA).

[1402] Compound administration and brain tissue collection. Wild-type C57BL / 6J male mice were obtained from Jackson laboratories (Bar Harbor, Maine). The acute single-dose effects of the Ca V 1.2 activators of the present disclosure were evaluated in 8-week-old male mice (n = 6 mice per compound). Each compound was dissolved in 10% PEG300, 10% Solutol, 10% Cremophore EL, and 70% phosphate-buffered saline and administered intraperitoneally (i.p.) at a concentration of 1 mg / kg to 30 mg / kg depending on the compound. Animals were euthanized by exsanguination under deep anesthesia one hour after compound administration. Blood was collected in EDTA tubes for downstream analysis of drug levels. Brains were rapidly removed from the skulls, and the cerebral cortex and cerebellum were dissected locally. Cerebellum samples were snap-frozen in liquid nitrogen to assess compound exposure. Cortex samples for cFos evaluation were placed in 500 μl of RNAlater solution (ThermoFisher) to maintain the integrity of RNA in the samples. Samples were kept in RNAlater at 4 °C for at least 24 hours and then moved to -80 °C for storage prior to processing.

[1403] Quantify cFos mRNA induction. Tissue homogenization was performed using a TissueLyser system (Qiagen) in 96-well plates. First, the frozen cortical samples were thawed, removed from RNAlater, and placed into TissueLyser tubes together with buffer RLT containing 0.5% reagent DX and a 5 mm TissueLyser metal bead. The TissueLyser tubes were loaded into a TissueLyser II tissue homogenizer for 3 rounds of homogenization, with each round of homogenization lasting 5 minutes at a bead milling frequency of 30 Hz. Total RNA was purified from the homogenate using an RNeasy 96 Plus kit (Qiagen), and the RNA concentration and A260 / A280 ratio were quantified by Nanodrop (Thermo Fisher Scientific), and all samples were normalized relative to a concentration of 100 ng / μL. RNA was reverse transcribed into cDNA using a Superscript III First-strand synthesis SuperMix Kit (Thermo Fisher Scientific). For each sample, 6 μL of RNA (600 ng total) was mixed with 1 μL of oligo dT and 1 μL of annealing buffer and heated to 65 °C for 5 minutes. Next, 10 μL of 2X First-Strand Reaction Mix and 2 μL of enzyme mix were added to make a total reaction volume of 20 μL. The samples were heated to 50 °C for 50 minutes and then to 85 °C for 5 minutes to complete cDNA synthesis.

[1404] Quantitative PCR was performed on the cDNA samples in 384-well assay format using a Quantitect Multiplex RT-PCR kit (Qiagen). Each PCR well contained 2 μl of cDNA (60 ng total), 10 μL of RT-PCR premix, 1 μL of cFos FAM Taqman probe (Mm00487425_m1 (FAM) #4351368), 1 μL of GAPDH VIC Taqman probe (Mm99999915-g1 (VIC) #4448486), 0.2 μl of Multiplex RT mix, and 5.8 μl of RNase-free water. On a ViiA7 real-time PCR system (Thermo Fisher Scientific), the samples were heated to 95 °C for 15 minutes and then cycled between 94 °C for 45 seconds and 60 °C for 45 seconds for 45 cycles. The cFos Ct values were exported, normalized relative to the GAPDH Ct values, and converted to relative fold changes in expression using the Δ-ΔCt relative quantification method. The cFos fold changes between compound treatment and vehicle were analyzed by one-way ANOVA, followed by Tukey's post hoc comparison.

[1405] Quantify compound exposure. For every 1 g of tissue, homogenize cerebellar tissue samples in 4 ml of 20% acetonitrile and 80% phosphate buffered saline (5x dilution). Depending on the tissue type, homogenize the tissue using one of the following three methods: hand-held probe system, tissue grinder system with 5 mm steel beads, for 30 s -1 , for 4 min, or OMNI Bead Ruptor Elite homogenizer, for 30 seconds to 1 minute. Add the tissue samples to a 96-well plate (12.5 uL sample) and process for quantification by mass spectrometry.

[1406] Results

[1407] Table 9: Compound data

[1408]

[1409]

[1410] Qpatch is performed at 6 dose responses with 10 individual measurements at each concentration. Unless stated otherwise there is only one experimental repeat (n = 1)

[1411] *n = 2

[1412] #n = 3

[1413] nd = not determined

[1414] Comparative Example

[1415] Other Ca V 1.2 activators are known, however these compounds are less effective and / or do not have the desired biophysical properties required to activate the channel and do not have sufficient brain exposure while minimizing cardiovascular risks (such as increased blood pressure, altered heart rate or contractility and / or arrhythmias due to QT prolongation).

[1416] Table 10. Comparative examples with other known CaV1.2 activators

[1417]

[1418]

Claims

1. Methyl (R)-4-(3-fluoro-2-((R)-1-fluoroethyl)phenyl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the treatment of schizophrenia.

2. Use according to claim 1, wherein the compound is in crystalline form.

3. The use according to claim 2, wherein the crystalline form is Form A, characterized in that Having an X-ray powder diffraction pattern with reflections at the following 2-theta angles: 8.5° ± 0.2, 10.4° ± 0.2 and 10.8° ± 0.

2.

4. Use according to claim 2, wherein the crystalline form is Form B, characterized in that Having an X-ray powder diffraction pattern with reflections at the following 2-theta angles: 12.0° ± 0.2, 13.7° ± 0.2 and 21.4° ± 0.

2.

5. The use according to claim 2, wherein the crystalline form is Form C, characterized in that Having an X-ray powder diffraction pattern with reflections at the following 2-theta angles: 10.3° ± 0.2, 15.5° ± 0.2° and 20.6° ± 0.2.

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